Glycan-dependent leukocyte adhesion and recruitment in inflammation

Leukocyte recruitment in acute and chronic inflammation is characterized by sequential cell adhesion and activation events. E-, P- and L-selectins mediate initial leukocyte-endothelial-cell adhesion events required for this process. Each selectin recognizes related but distinct counter-receptors displayed by leukocytes and/or the endothelium. These counter-receptors correspond to specific glycoproteins whose 'activity' is enabled by carefully controlled post-translational modifications. Characterization of the glycans associated with E- and P-selectin counter-receptors, and of mice with targeted deletions of glycosyltransferase and sulfotransferase genes, disclose that neutrophil E- and/or P-selectin counter-receptor activities derive, minimally, from essential synthetic collaborations amongst polypeptide N-acetylgalactosaminyltransferase(s), a beta-N-acetylglucosaminyltransferase that assembles core-2-type O-glycans, beta-1,4-galactosyltransferase(s), protein tyrosine sulfotransferase(s), alpha-2,3-sialyltransferases, and a pair of alpha-1,3-fucosyltransferases.     

N-Glycans differentially regulate eosinophil and neutrophil recruitment during allergic airway inflammation

Allergic airway inflammation, including asthma, is usually characterized by the predominant recruitment of eosinophils. However, neutrophilia is also prominent during severe exacerbations. Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment during inflammation. Here, the involvement of UDP-N-acetylglucosamine:α-6-D-mannoside β1,6-N-acetylglucosaminyltransferase V (MGAT5)-modified N-glycans in eosinophil and neutrophil recruitment during allergic airway inflammation was investigated. Allergen-challenged Mgat5-deficient (Mgat5(-/-)) mice exhibited significantly attenuated airway eosinophilia and inflammation (decreased Th2 cytokines, mucus production) compared with WT counterparts, attributable to decreased rolling, adhesion, and survival of Mgat5(-/-) eosinophils. Interestingly, allergen-challenged Mgat5(-/-) mice developed airway neutrophilia and increased airway reactivity with persistent elevated levels of proinflammatory cytokines (IL-17A, TNFα, IFNγ)). This increased neutrophil recruitment was also observed in LPS- and thioglycollate (TG)-induced inflammation in Mgat5(-/-) mice. Furthermore, there was significantly increased recruitment of infused Mgat5(-/-) neutrophils compared with WT neutrophils in the peritoneal cavity of TG-exposed WT mice. Mgat5(-/-) neutrophils demonstrated enhanced adhesion to P-selectin as well as increased migration toward keratinocyte-derived chemokine compared with WT neutrophils in vitro along with increased calcium mobilization upon activation and expression of elevated levels of CXCR2, which may contribute to the increased neutrophil recruitment. These data indicate an important role for MGAT5-modified N-glycans in differential regulation of eosinophil and neutrophil recruitment during allergic airway inflammation.     

Role of interleukin-1 and tumour necrosis factor in leukocyte recruitment to acute dermal inflammation

The cytokines IL-1 and TNF-alpha are involved in inflammation and their production is stimulated by various agents, especially endotoxin (LPS). Here, using the human IL-1 receptor antagonist (IL-1RA) and a new monoclonal antibody (mAb 7F11) to rabbit TNF, the role of endogenous IL-l and TNF production in acute (3h) leukocyte (PMNL) recruitment to dermal inflammation in rabbits has been studied. IL-1RA inhibited by 27% the PMNL accumulation in reactions induced by killed Escherichia coli (p < 0.05) but not by LPS. The monoclonal antibody to TNF inhibited by 27% and 38% (p < 0.002) the PMNL accumulation in LPS and E. coli reactions respectively, but a combination of the mAb with IL-1RA was not more effective. Treatment of human umbilical vein endothelium with LPS for 3 h activated endothelium to induce PMNL transendothelial migration in vitro, which was not inhibited by IL-1RA, antibody to TNF-alpha, IL-1 or to IL-8. In conclusion, TNF and IL-1 may partially mediate acute PMNL infiltration in vivo to LPS and Gram negative bacteria, but there is a major IL-1/TNF independent mechanism, at least in dermal inflammation, which may be due to direct LPS activation of the microvasculature or perhaps the generation of cytokines other than IL-1 and TNF.     

Blockade of CD49d inhibits allergic airway pathologies independent of effects on leukocyte recruitment

Lymphocyte and/or eosinophil recruitment is dependent on the sequential interactions between adhesion molecules expressed on activated endothelial cells and both leukocyte subtypes. Endothelial P- and E-selectins mediate tethering and rolling of leukocytes through interactions with P-selectin glycoprotein ligand-1 (PSGL-1), and diapedesis subsequently occurs by engagement of endothelial vascular cell adhesion molecule-1 and CD49d (alpha(4)-integrins). The anti-inflammatory potential of interfering with these adhesive interactions was assessed with an ovalbumin challenge mouse model of asthma. Administration of a soluble form of PSGL-1 reduced eosinophils (80%) and lymphocytes (50%) in bronchoalveolar lavage fluid without affecting epithelial changes or airway hyperreactivity (AHR). In contrast, although administration of anti-CD49d monoclonal antibodies (PS/2) resulted in similar reductions in eosinophils (75%) and lymphocytes (50%), PS/2 reduced and abolished mucous cell metaplasia and AHR, respectively. Administration of both PSGL-1 and PS/2 had the additive effect of eliminating eosinophils from the airways (96% decrease), with few or no additional reductions (relative to PS/2 administration alone) in lymphocyte recruitment, mucous cell metaplasia, or AHR. These data show that eosinophils and lymphocytes differentially utilize adhesive interactions during recruitment and that the inhibition of AHR is independent of this recruitment.     

Selective recruitment of Nck and Syk contribute to distinct leukocyte immune-type receptor-initiated target interactions

The ability of phagocytes to recognize, immobilize, and engulf extracellular targets are fundamental immune cell processes that allow for the destruction of a variety of microbial intruders. The phagocytic process depends onsignalling events that initiate dynamic changes in the plasma membrane architecture that are required to accommodate the internalization of large particulate targets. To better understand fundamental molecular mechanisms responsible for facilitating phagocytic receptor-mediated regulation of cytoskeletal networks, our research has focused on investigating representative immunoregulatory proteins from the channel catfish (Ictalurus punctatus) leukocyte immune-type receptor family (IpLITRs). Specifically, we have shown that a specific IpLITR-type can regulate the constitutive deployment of filopodial-like structures to actively capture and secure targets to the phagocyte surface, which is followed by F-actin mediated membrane dynamics that are associated with the formation of phagocytic cup-like structures that precede target engulfment. In the present study, we use confocal imaging to examine the recruitment of mediators of the F-actin cytoskeleton during IpLITR-mediated regulation of membrane dynamics. Our results provide novel details regarding the dynamic recruitment of the signaling effectors Nck and Syk during classical as well as atypical IpLITR-induced phagocytic processes.     

Decreased leukocyte recruitment by inorganic nitrate and nitrite in microvascular inflammation and NSAID-induced intestinal injury

Nitric oxide (NO) generated by vascular NO synthases can exert anti-inflammatory effects, partly through its ability to decrease leukocyte recruitment. Inorganic nitrate and nitrite, from endogenous or dietary sources, have emerged as alternative substrates for NO formation in mammals. Bioactivation of nitrate is believed to require initial reduction to nitrite by oral commensal bacteria. Here we investigated the effects of inorganic nitrate and nitrite on leukocyte recruitment in microvascular inflammation and in NSAID-induced small-intestinal injury. We show that leukocyte emigration in response to the proinflammatory chemokine MIP-2 is reduced by 70% after 7 days of dietary nitrate supplementation as well as by acute intravenous nitrite administration. Nitrite also reduced leukocyte adhesion to a similar extent and this effect was inhibited by the soluble guanylyl cyclase inhibitor ODQ, whereas the effect on emigrated leukocytes was not altered by this treatment. Further studies in TNF-α-stimulated endothelial cells revealed that nitrite dose-dependently reduced the expression of ICAM-1. In rats and mice subjected to a challenge with diclofenac, dietary nitrate prevented the increase in myeloperoxidase and P-selectin levels in small-intestinal tissue. Antiseptic mouthwash, which eliminates oral nitrate reduction, markedly blunted the protective effect of dietary nitrate on P-selectin levels. Despite attenuation of the acute immune response, the overall ability to clear an infection with Staphylococcus aureus was not suppressed by dietary nitrate as revealed by noninvasive IVIS imaging. We conclude that dietary nitrate markedly reduces leukocyte recruitment to inflammation in a process involving attenuation of P-selectin and ICAM-1 upregulation. Bioactivation of dietary nitrate requires intermediate formation of nitrite by oral nitrate-reducing bacteria and then probably further reduction to NO and other bioactive nitrogen oxides in the tissues.     

Alpha 4 integrin-dependent leukocyte recruitment does not require VCAM-1 in a chronic model of inflammation

Rats immunized with Mycobacterium butyricum in Freund's adjuvant develop a chronic vasculitis, with large increases in leukocyte rolling and adhesion in mesenteric postcapillary venules that are significantly inhibited with an alpha 4 integrin Ab. Using intravital microscopy to visualize chronically inflamed microvessels, we demonstrated that alpha 4 integrin-dependent leukocyte rolling and adhesion was inhibited with a beta 1 integrin, but not a beta 7 integrin Ab. To date, VCAM-1 has been presumed to be the primary ligand for alpha 4 beta 1 integrin in the vasculature. However, alpha 4 beta 1 integrin-dependent interactions were not reduced by monoclonal or polyclonal VCAM-1 Abs or a VCAM-1 antisense oligonucleotide despite increased VCAM-1 expression in the mesenteric vasculature. To ensure that the VCAM-1 Abs were functional and used at saturating concentrations, blood from Ab-treated rats was perfused over monolayers of CHO cells transfected with rat VCAM-1. Sufficient alpha 4 integrin or VCAM-1 Ab was present to inhibit leukocyte interactions with rat VCAM-1 by 95-100%. Under in vitro flow conditions, only mononuclear leukocytes were recruited from blood of control rats onto purified VCAM-1. However, neutrophils were also recruited onto VCAM-1 from whole blood of adjuvant-immunized animals via alpha 4 integrin. Another ligand for alpha 4 beta 1 integrin is the connecting segment-1 (CS-1) region of fibronectin. An Ab to the CS-1 portion of fibronectin, which did not reduce rolling and adhesion in adjuvant arthritis animals, completely inhibited leukocyte adhesion to CS-1 under static conditions. These findings provide the first evidence that alpha 4 beta 1 integrin-dependent leukocyte rolling and adhesion can occur in vivo via a mechanism other than VCAM-1.     

Thrombin and leukocyte recruitment in endotoxemia

Because thrombin has been implicated in sepsis, it has been proposed that antithrombin III (AT III) is beneficial due to its anticoagulatory and antiadhesive effects. Using intravital microscopy, we visualized leukocyte-endothelium interactions in postcapillary venules of the feline mesentery exposed to lipopolysaccharide (LPS). At a concentration of AT III that blocks leukocyte adhesion in postischemic mesentery, we found no role for thrombin in LPS-induced rolling, adhesion and emigration, or microvascular dysfunction. Furthermore, AT III did not attenuate leukocyte-endothelial interactions after tumor necrosis factor-alpha superfusion of the mesentery. In contrast, fucoidan, a selectin inhibitor, prevented almost all LPS-induced rolling and reduced adhesion, emigration, and microvascular dysfunction. In a model of endotoxemia, leukocyte recruitment into mesentery or lungs was unaffected by AT III. Finally, in a human cell system that mimics the flow conditions in vivo, human neutrophils rolled, adhered, and emigrated similar to the feline postcapillary microvessels, and AT III had no effect on leukocyte recruitment induced by LPS. If AT III has beneficial effects in endotoxemia, it is not due to a direct effect upon leukocyte rolling, adhesion, or emigration in postcapillary venules in vivo.     

Role of integrin-linked kinase in leukocyte recruitment

Chemokines modulate leukocyte integrin avidity to coordinate adhesion and subsequent transendothelial migration, although the sequential signaling pathways involved remain poorly characterized. Here we show that integrin-linked kinase (ILK), a 59-kDa serine-threonine protein kinase that interacts principally with beta(1) integrins, is highly expressed in human mononuclear cells and is activated by exposure of leukocytes to the chemokine monocyte chemoattractant protein-1. Biochemical inhibitor studies show that chemokine-triggered activation of ILK is downstream of phosphoinositide 3-kinase. In functional assays under physiologically relevant flow conditions, overexpression of wild-type ILK in human monocytic cells diminishes beta(1) integrin/vascular cell adhesion molecule-1-dependent firm adhesion to human endothelial cells. These data implicate ILK in the dynamic signaling events involved in the regulation of leukocyte integrin avidity for endothelial substrates.     

Resident cell chemokine expression serves as the major mechanism for leukocyte recruitment during local inflammation

The mechanistic relationships between initiating stimulus, cellular source and sequence of chemokine expression, and leukocyte recruitment during inflammation are not clear. To study these relationships in an acute inflammatory process, we challenged a murine air pouch with carrageenan. A time-dependent increase in TNF-alpha, monocyte chemottractant protein-1 (MCP-1), macrophage-inflammatory protein-1alpha (MIP-1alpha), RANTES, KC, and MIP-2 was found in the exudates preceding cell recruitment, but displaying different kinetic profiles. Air pouches generated for 2, 6, or 9 days before initiating inflammation demonstrated a proportional increase in the number of cells lining the cavities. Two hours after carrageenan stimulation, the synthesis of TNF-alpha and all chemokines but RANTES increased in proportion to the lining cellularity, although no differences in infiltrating leukocytes were found, suggesting that the early source of these mediators is resident cells. To assess the contribution of neutrophils to chemokine synthesis at later time points, we used neutropenic animals. Neutrophil depletion caused a decrease in TNF-alpha (51%), KC (37%), MIP-1alpha (30%), and RANTES (57%) levels and a 2-fold increase in monocytes 4 h after challenge. No effect on MIP-2 and MCP-1 levels was observed. The selective blockade of CXCR2 or CCR1 inhibited neutrophil recruitment by 74% and 54%, respectively, without a significant inhibition of monocytes. A differential effect on TNF-alpha and MCP-1 levels was observed after these treatments, indicating that the two receptors did not subserve a mere redundant chemotactic role. Overall, our results suggest that chemokines synthesized by resident cells play an important role in the evolution of the inflammatory response.     

SG-HQ2 inhibits mast cell-mediated allergic inflammation through suppression of histamine release and pro-inflammatory cytokines

In this study, we investigated the effect of 3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide) (SG-HQ2), a synthetic analogue of gallic acid (3,4,5-trihydroxybenzoic acid), on the mast cell-mediated allergic inflammation and the possible mechanism of action. Mast cells play major roles in immunoglobulin E-mediated allergic responses by the release of histamine, lipid-derived mediators, and pro-inflammatory cytokines. We previously reported the potential effects of gallic acid using allergic inflammation models. For incremental research, we synthesized the SG-HQ2 by the modification of functional groups from gallic acid. SG-HQ2 attenuated histamine release by the reduction of intracellular calcium in human mast cells and primary peritoneal mast cells. The inhibitory efficacy of SG-HQ2 was similar with gallic acid. Enhanced expression of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, interleukin-4, and interleukin-6 in activated mast cells was significantly diminished by SG-HQ2 100 times lower concentration of gallic acid. This inhibitory effect was mediated by the reduction of nuclear factor-κB. In animal models, SG-HQ2 inhibited compound 48/80-induced serum histamine release and immunoglobulin E-mediated local allergic reaction, passive cutaneous anaphylaxis. Our results indicate that SG-HQ2, an analogue of gallic acid, might be a possible therapeutic candidate for mast cell-mediated allergic inflammatory diseases through suppression of histamine release and pro-inflammatory cytokines.     

Eosinophil tissue recruitment to sites of allergic inflammation in the lung is platelet endothelial cell adhesion molecule independent

Platelet endothelial cell adhesion molecule (PECAM or CD31) is a cell adhesion molecule expressed on circulating leukocytes and endothelial cells that plays an important role in mediating neutrophil and monocyte transendothelial migration in vivo. In this study, we investigated whether eosinophils, like neutrophils and monocytes, utilize PECAM for tissue recruitment to sites of allergic inflammation in vivo. Eosinophils express similar levels of PECAM as neutrophils as assessed by FACS analysis. RT-PCR studies demonstrate that eosinophils like neutrophils express the six extracellular domains of PECAM. Eosinophils exhibit homophilic binding to recombinant PECAM as assessed in a single-cell micropipette adhesion assay able to measure the biophysical strength of adhesion of eosinophils to recombinant PECAM. The strength of eosinophil adhesion to recombinant PECAM is the same as that of neutrophil binding to recombinant PECAM and can be inhibited with an anti-PECAM Ab. Although eosinophils express functional PECAM, anti-PECAM Abs did not inhibit bronchoalveolar lavage eosinophilia, lung eosinophilia, and airway hyperreactivity to methacholine in a mouse model of OVA-induced asthma in vivo. Thus, in contrast to studies that have demonstrated that neutrophil and monocyte tissue recruitment is PECAM dependent, these studies demonstrate that eosinophil tissue recruitment in vivo in this model is PECAM independent.     

Hepatic leukocyte recruitment in a model of acute colitis

There is a close relationship between inflammatory bowel disease (IBD) and various hepatobiliary disorders. The objective of this study was to determine whether hepatic leukocyte recruitment occurs in experimental colitis. We used the murine model of colitis induced by 2,4-dinitrobenezenesulfonic acid (DNBS). Male C57Bl/6 mice received an intrarectal injection of 4 mg DNBS in 100 microl 50% ethanol. Controls received 100 microl 50% ethanol. The hepatic microcirculation was examined at 3 and 14 days post-DNBS by intravital video microscopy. Three days post-DNBS, when mice had developed acute colitis, there was associated hepatic leukocyte recruitment. Within the postsinusoidal venules there was a fourfold increase in the flux of rolling leukocytes that was P-selectin dependent but not alpha(4)-integrin dependent. There was also an increase in stationary leukocytes within the sinusoids, although this was not associated with an increase in serum alanine transaminase. By 14 days post-DNBS when macroscopic evidence of colonic inflammation was resolved, rolling within the postsinusoidal venules had returned to control levels. In this murine model of colitis, we describe a link between acute colonic inflammation and remote hepatic leukocyte recruitment that is P-selectin dependent. Active IBD may lead to remote hepatic inflammation.     

Molecular mechanisms of leukocyte recruitment in postischemic liver microcirculation

Evidence shows that leukocyte recruitment into inflamed liver sinusoids does not require selectins, with one notable exception: ischemia-reperfusion (I/R). We used intravital microscopy to directly visualize the liver microcirculation during I/R and localized endotoxemia (liver superfused with lipopolysaccharide). General anti-selectin therapy (fucoidan) or anti-adhesion therapy with an antithrombin inhibitor (hirudin) was also used. Many neutrophils rolled and adhered in postsinusoidal vessels and sequestered in the sinusoids during I/R and local endotoxin superfusion. Although fucoidan blocked rolling in both forms of inflammation, leukocyte recruitment into sinusoids was only blocked in I/R. Adhesion was also inhibited in postischemic sinusoids with a second anti-adhesive agent (hirudin). Because liver I/R inevitably induces ischemia upstream in the intestine, anti-selectin therapy may prevent intestinal injury, which could prevent downstream liver inflammation. To test this hypothesis, we completely removed the intestine and rerouted blood flow from the superior mesenteric artery to the superior mesenteric vein. I/R was induced in the liver microcirculation, and many leukocytes rolled and adhered in postsinusoidal venules and adhered in sinusoids. Although fucoidan significantly reduced the rolling in postsinusoidal vessels, adhesion persisted in the sinusoids. Our data suggest that anti-adhesion therapy is effective in liver I/R in the sinusoids and postsinusoidal venules, perhaps in part due to its beneficial effect on the intestine.     

Mechanisms of platelet and leukocyte recruitment in experimental colitis

Both leukocytes and platelets accumulate in the colonic microvasculature during experimental colitis, leading to microvascular dysfunction and tissue injury. The objective of this study was to determine whether the recruitment of leukocytes and platelets in inflamed colonic venules are codependent processes. The rolling and adherence of leukocytes and platelets in colonic venules of mice with dextran sodium sulfate (DSS)-induced colitis were monitored by intravital videomicroscopy. DSS elicited an increased recruitment of both rolling and adherent leukocytes and platelets. DSS-colitic mice rendered thrombocytopenic with anti-platelet serum exhibited profound reductions in leukocyte adhesion. Neutropenia, induced with anti-neutrophil serum, significantly reduced the adhesion of leukocytes and the accumulation of platelet-leukocyte aggregates while greatly enhancing the number of platelets that roll and adhere directly to venular endothelial cells. The enhanced platelet adhesion associated with neutropenia was mediated by platelet P-selectin interactions with endothelial cell P-selectin glycoprotein ligand (PSGL-1). DSS colitis was also associated with an increased expression of PSGL-1 in the colonic vasculature. These findings indicate that the recruitment of leukocytes and platelets in inflamed colonic venules are co-dependent processes.     

Selective suppression of adipose tissue apoE expression impacts systemic metabolic phenotype and adipose tissue inflammation

apoE is a multi-functional protein expressed in several cell types and in several organs. It is highly expressed in adipose tissue, where it is important for modulating adipocyte lipid flux and gene expression in isolated adipocytes. In order to investigate a potential systemic role for apoE that is produced in adipose tissue, mice were generated with selective suppression of adipose tissue apoE expression and normal circulating apoE levels. These mice had less adipose tissue with smaller adipocytes containing fewer lipids, but no change in adipocyte number compared with control mice. Adipocyte TG synthesis in the presence of apoE-containing VLDL was markedly impaired. Adipocyte caveolin and leptin gene expression were reduced, but adiponectin, PGC-1, and CPT-1 gene expression were increased. Mice with selective suppression of adipose tissue apoE had lower fasting lipid, insulin, and glucose levels, and glucose and insulin tolerance tests were consistent with increased insulin sensitivity. Lipid storage in muscle, heart, and liver was significantly reduced. Adipose tissue macrophage inflammatory activation was markedly diminished with suppression of adipose tissue apoE expression. Our results establish a novel effect of adipose tissue apoE expression, distinct from circulating apoE, on systemic substrate metabolism and adipose tissue inflammatory state.     

Nox enzymes in allergic airway inflammation

Chronic airway diseases such as asthma are linked to oxidative environmental factors and are associated with increased production of reactive oxygen species (ROS). Therefore, it is commonly assumed that oxidative stress is an important contributing factor to asthma disease pathogenesis and that antioxidant strategies may be useful in the treatment of asthma. A primary source of ROS production in biological systems is NADPH oxidase (NOX), originally associated primarily with inflammatory cells but currently widely appreciated as an important enzyme system in many cell types, with a wide array of functional properties ranging from antimicrobial host defense to immune regulation and cell proliferation, differentiation and apoptosis. Given the complex nature of asthma disease pathology, involving many lung cell types that all express NOX homologs, it is not surprising that the contributions of NOX-derived ROS to various aspects of asthma development and progression are highly diverse and multifactorial. It is the purpose of the present review to summarize the current knowledge with respect to the functional aspects of NOX enzymes in various pulmonary cell types, and to discuss their potential importance in asthma pathogenesis. This article is part of a Special Issue entitled: Biochemistry of Asthma.     

Dendritic cells in allergic airway inflammation

Dendritic cells (DCs) are primary antigen-presenting cells involved in interactions with T cells leading to the proliferation of TH1 or TH2 cell types. In asthma, predominance of TH2 cells appears to be responsible for disease pathogenesis. Differentiation of TH2 cells is driven by a variety of factors such as the expression of high levels of costimulatory molecules, the cytokine profile, and the subset of DCs. Many inflammatory cells involved in the pathogenesis of asthma either directly or indirectly modulate DC function. Traditional treatments for asthma decrease the number of airway DCs in animals as well as in patients with asthma. Immunomodulators including interleukin (IL)-10, transforming growth factor (TGF)-beta, cytosine-phosphate-guanosine-containing oligodeoxynucleotides (CpG-ODN), 1alpha,25-dihydroxyvitamin D3, and fetal liver tyrosine kinase 3 ligand (Flt3L) are involved in the modulation of the function of DCs. Based on the critical review of the interaction between DCs and other inflammatory cells, we propose that activation of T cells by DCs and sensitization to inhaled allergen and resulting airway inflammation are dependent on plasmacytoid and myeloid subset of lung DCs to induce an immune response or tolerance and are tightly regulated by T-regulatory cells. Effects of various therapeutic agents to modulate the function of lung myeloid DCs have been discussed.