CD103 Regulates Dermal Regulatory T Cell Motility and Interactions with CD11c-Expressing Leukocytes to Control Skin Inflammation.

Dermal regulatory T cells (Tregs) are essential for maintenance of skin homeostasis and control of skin inflammatory responses. In mice, Tregs in the skin are characterized by high expression of CD103, the αE integrin. Evidence indicates that CD103 promotes Treg retention within the skin, although the mechanism underlying this effect is unknown. The main ligand of CD103, E-cadherin, is predominantly expressed by cells in the epidermis. However, because Tregs are predominantly located within the dermis, the nature of the interactions between E-cadherin and CD103-expressing Tregs is unclear. In this study, we used multiphoton intravital microscopy to examine the contribution of CD103 to Treg behavior in resting and inflamed skin of mice undergoing oxazolone-induced contact hypersensitivity. Inhibition of CD103 in uninflamed skin did not alter Treg behavior, whereas 48 h after inducing contact hypersensitivity by oxazolone challenge, CD103 inhibition increased Treg migration. This coincided with E-cadherin upregulation on infiltrating myeloid leukocytes in the dermis. Using CD11c-enhanced yellow fluorescent protein (EYFP) × Foxp3-GFP dual-reporter mice, inhibition of CD103 was found to reduce Treg interactions with dermal dendritic cells. CD103 inhibition also resulted in increased recruitment of effector CD4+ T cells and IFN-γ expression in challenged skin and resulted in reduced glucocorticoid-induced TNFR-related protein expression on Tregs. These results demonstrate that CD103 controls intradermal Treg migration, but only at later stages in the inflammatory response, when E-cadherin expression in the dermis is increased, and provide evidence that CD103-mediated interactions between Tregs and dermal dendritic cells support regulation of skin inflammation.

Lung Imaging Reveals Stroke-Induced Impairment in Pulmonary Intravascular Neutrophil Function, a Response Exacerbated with Aging.

In stroke patients, infection is a significant contributor to morbidity and mortality. Moreover, older stroke patients show an increased risk of developing stroke-associated infection, although the mechanisms underlying this increased susceptibility to infection are unknown. In this study, using an experimental mouse model of ischemic stroke, we showed that older (12-15 mo of age) mice had elevated lung bacterial infection and inflammatory damage after stroke when compared with young (8-10 wk of age) counterparts, despite undergoing the same degree of brain injury. Intravital microscopy of the lung microvasculature revealed that in younger mice, stroke promoted neutrophil arrest in pulmonary microvessels, but this response was not seen in older poststroke mice. In addition, bacterial phagocytosis by neutrophils in the lung microvasculature was reduced by both aging and stroke, such that neutrophils in aged poststroke mice showed the greatest impairment in this function. Analysis of neutrophil migration in vitro and in the cremaster muscle demonstrated that stroke alone did not negatively impact neutrophil migration, but that the combination of increased age and stroke led to reduced effectiveness of neutrophil chemotaxis. Transcriptomic analysis of pulmonary neutrophils using RNA sequencing identified 79 genes that were selectively altered in the context of combined aging and stroke, and they were associated with pathways that control neutrophil chemotaxis. Taken together, the findings of this study show that stroke in older animals results in worsening of neutrophil antibacterial responses and changes in neutrophil gene expression that have the potential to underpin elevated risk of stroke-associated infection in the context of increased age.

Removal of the endothelial surface layer via hyaluronidase does not modulate monocyte and neutrophil interactions with the glomerular endothelium.

OBJECTIVE: The endothelial surface layer (ESL), a layer of macromolecules on the surface of endothelial cells, can both impede and facilitate leukocyte recruitment. However, its role in monocyte and neutrophil recruitment in glomerular capillaries is unknown. METHODS: We used multiphoton intravital microscopy to examine monocyte and neutrophil behavior in the glomerulus following ESL disruption with hyaluronidase. RESULTS: Constitutive retention and migration of monocytes and neutrophils within the glomerular microvasculature was unaltered by hyaluronidase. Consistent with this, inhibition of the hyaluronan-binding molecule CD44 also failed to modulate glomerular trafficking of these immune cells. To investigate the contribution of the ESL during acute inflammation, we induced glomerulonephritis via in situ immune complex deposition. This resulted in increases in glomerular retention of monocytes and neutrophils but did not induce marked reduction in the glomerular ESL. Furthermore, hyaluronidase treatment did not modify the prolonged retention of monocytes and neutrophils in the acutely inflamed glomerular microvasculature. CONCLUSIONS: These observations indicate that, despite evidence that the ESL has the capacity to inhibit leukocyte-endothelial cell interactions while also containing adhesive ligands for immune cells, neither of these functions modulate trafficking of monocytes and neutrophils in steady-state or acutely-inflamed glomeruli.

Leukocyte Tetraspanin CD53 Restrains α3 Integrin Mobilization and Facilitates Cytoskeletal Remodeling and Transmigration in Mice.

The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53-/- mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53-/- neutrophils revealed no alteration in expression of ß2 integrins, whereas L-selectin was almost completely absent from Cd53-/- neutrophils. In addition, Cd53-/- neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased α3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53-/- mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function.

Tetraspanin CD53 modulates lymphocyte trafficking but not systemic autoimmunity in Lyn-deficient mice.

The leukocyte-restricted tetraspanin CD53 has been shown to promote lymphocyte homing to lymph nodes (LNs) and myeloid cell recruitment to acutely inflamed peripheral organs, and accelerate the onset of immune-mediated disease. However, its contribution in the setting of chronic systemic autoimmunity has not been investigated. We made use of the Lyn-/- autoimmune model, generating Cd53-/- Lyn-/- mice, and compared trafficking of immune cells into secondary lymphoid organs and systemic autoimmune disease development with mice lacking either gene alone. Consistent with previous observations, absence of CD53 led to reduced LN cellularity via reductions in both B and T cells, a phenotype also observed in Cd53-/- Lyn-/- mice. In some settings, Cd53-/- Lyn-/- lymphocytes showed greater loss of surface L-selectin and CD69 upregulation above that imparted by Lyn deficiency alone, indicating that absence of these two proteins can mediate additive effects in the immune system. Conversely, prototypical effects of Lyn deficiency including splenomegaly, plasma cell expansion, elevated serum immunoglobulin M and anti-nuclear antibodies were unaffected by CD53 deficiency. Furthermore, while Lyn-/- mice developed glomerular injury and showed elevated glomerular neutrophil retention above than that in wild-type mice, absence of CD53 in Lyn-/- mice did not alter these responses. Together, these findings demonstrate that while tetraspanin CD53 promotes lymphocyte trafficking into LNs independent of Lyn, it does not make an important contribution to development of autoimmunity, plasma cell dysfunction or glomerular injury in the Lyn-/- model of systemic autoimmunity.

Platelet retention in inflamed glomeruli occurs via selective prolongation of interactions with immune cells.

Platelet-leukocyte interactions promote acute glomerulonephritis. However, neither the nature of the interactions between platelets and immune cells nor the capacity of platelets to promote leukocyte activation has been characterized in this condition. We used confocal intravital microscopy to define the interactions of platelets with neutrophils, monocytes, and endothelial cells in glomerular capillaries in mice. In the absence of inflammation, platelets underwent rapid on/off interactions with immune cells. During glomerulonephritis induced by in situ immune complex formation, platelets that interacted with neutrophils or monocytes, but not with other intraglomerular cells, were retained in the glomerulus for prolonged durations. Depletion of platelets inhibited both neutrophil recruitment and activation. Inhibition of platelet activating factor reduced neutrophil recruitment without impacting reactive oxygen species generation, while blocking CXC chemokine ligand 7 (CXCL7) reduced both responses. In contrast, inhibition of the adenosine diphosphate and thromboxane A2 pathways inhibited neutrophil reactive oxygen species generation without affecting neutrophil adhesion. Thus, platelet retention in glomerular capillaries following immune complex deposition stems from prolongation of platelet interactions with immune cells but not other substrates. Pro-inflammatory mediators play divergent roles in promoting neutrophil retention and activation in glomerular capillaries.

Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus.

Nonclassical monocytes undergo intravascular patrolling in blood vessels, positioning them ideally to coordinate responses to inflammatory stimuli. Under some circumstances, the actions of monocytes have been shown to involve promotion of neutrophil recruitment. However, the mechanisms whereby patrolling monocytes control the actions of neutrophils in the circulation are unclear. Here, we examined the contributions of monocytes to antibody- and neutrophil-dependent inflammation in a model of in situ immune complex-mediated glomerulonephritis. Multiphoton and spinning disk confocal intravital microscopy revealed that monocytes patrol both uninflamed and inflamed glomeruli using ß2 and α4 integrins and CX3CR1. Monocyte depletion reduced glomerular injury, demonstrating that these cells promote inappropriate inflammation in this setting. Monocyte depletion also resulted in reductions in neutrophil recruitment and dwell time in glomerular capillaries and in reactive oxygen species (ROS) generation by neutrophils, suggesting a role for cross-talk between monocytes and neutrophils in induction of glomerulonephritis. Consistent with this hypothesis, patrolling monocytes and neutrophils underwent prolonged interactions in glomerular capillaries, with the duration of these interactions increasing during inflammation. Moreover, neutrophils that interacted with monocytes showed increased retention and a greater propensity for ROS generation in the glomerulus. Also, renal patrolling monocytes, but not neutrophils, produced TNF during inflammation, and TNF inhibition reduced neutrophil dwell time and ROS production, as well as renal injury. These findings show that monocytes and neutrophils undergo interactions within the glomerular microvasculature. Moreover, evidence indicates that, in response to an inflammatory stimulus, these interactions allow monocytes to promote neutrophil recruitment and activation within the glomerular microvasculature, leading to neutrophil-dependent tissue injury.

C5a receptor 1 promotes autoimmunity, neutrophil dysfunction and injury in experimental anti-myeloperoxidase glomerulonephritis.

The prospects for complement-targeted therapy in ANCA-associated vasculitis have been enhanced by a recent clinical trial in which C5a receptor 1 (C5aR1) inhibition safely replaced glucocorticoids in induction treatment. C5aR1 primes neutrophils for activation by anti-neutrophil cytoplasmic antibody (ANCA) and is therefore required in models of glomerulonephritis induced by anti-myeloperoxidase antibody. Although humoral and cellular autoimmunity play essential roles in ANCA-associated vasculitis, a role for C5aR1 in these responses has not been described. Here, we use murine models to dissect the role of C5aR1 in the generation of anti-myeloperoxidase autoimmunity and the effector responses resulting in renal injury. The genetic absence or pharmacological inhibition of C5aR1 results in reduced autoimmunity to myeloperoxidase with an attenuated Th1 response, increased Foxp3+ regulatory T cells and reduction in generation of myeloperoxidase-ANCA. These changes are mediated by C5aR1 on dendritic cells, which promotes activation, and thus myeloperoxidase autoimmunity and glomerulonephritis. We also use renal intravital microscopy to determine the effect of C5aR1 inhibition on ANCA induced neutrophil dysfunction. We found that myeloperoxidase-ANCA induce neutrophil retention and reactive oxygen species burst within glomerular capillaries. These pathological behaviors are abrogated by C5aR1 inhibition. Thus, C5aR1 inhibition ameliorates both autoimmunity and intra-renal neutrophil activation in ANCA-associated vasculitis.

In Vivo Imaging of Inflamed Glomeruli Reveals Dynamics of Neutrophil Extracellular Trap Formation in Glomerular Capillaries.

Neutrophil extracellular traps (NETs) have been documented in glomeruli of patients with glomerulonephritis. However, the dynamics of NET formation in the glomerulus and their functional contribution to acute glomerular injury are poorly understood. Herein, we used in vivo multiphoton microscopy to investigate NET formation in the acutely inflamed glomerulus. Glomerular inflammation was induced using an antibody against the glomerular basement membrane. After induction of inflammation, multiphoton microscopy revealed that approximately 20% of glomeruli contained structures composed of extracellular DNA within the capillaries. These structures were not seen in mice depleted of neutrophils, consistent with them being NETs. Most contained myeloperoxidase, as seen in NETs in other tissues, whereas intraglomerular NETs did not contain significant levels of the histone H2Ax or neutrophil elastase. In vivo imaging revealed that intraglomerular NETs were present only transiently, suggesting that NETs were susceptible to disruption under the high shear conditions in glomerular capillaries. Investigation of NETs under flow conditions in vitro supported this concept. Dissolution of NETs via DNase I did not alter anti-glomerular basement membrane antibody-induced glomerular injury, as assessed via albuminuria, although the degree of microscopic hematuria was reduced by this intervention. These data indicate that in this model of acute, neutrophil-dependent glomerulonephritis, NETs are generated in the glomerular capillaries, where they are short lived and make a modest contribution to glomerular injury.

Tyrosine sulfation of chemokine receptor CCR2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein-1 (MCP-1).

Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1(T10C) was less effective at inducing leukocyte recruitment in vivo. In two-dimensional NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favor the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and ß3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N terminus of CCR2 destabilizes the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.

Macrophage migration inhibitory factor and CD74 regulate macrophage chemotactic responses via MAPK and Rho GTPase.

Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment to sites of inflammation. However, whether this stems from a direct effect on leukocyte migration is unknown. Furthermore, the role of the MIF-binding protein CD74 in this response has not been investigated. Therefore, the aim of this study was to examine the contributions of MIF and CD74 to chemokine-induced macrophage recruitment. Intravital microscopy studies demonstrated that CCL2-induced leukocyte adhesion and transmigration were reduced in MIF(-/-) and CD74(-/-) mice. MIF(-/-) and CD74(-/-) macrophages also exhibited reduced chemotaxis in vitro, although CD74(-/-) macrophages showed increased chemokinesis. Reduced CCL2-induced migration was associated with attenuated MAPK phosphorylation, RhoA GTPase activity, and actin polymerization in MIF(-/-) and CD74(-/-) macrophages. Furthermore, in MIF(-/-) macrophages, MAPK phosphatase-1 was expressed at elevated levels, providing a potential mechanism for the reduction in MAPK phosphorylation in MIF-deficient cells. No increase in MAPK phosphatase-1 expression was observed in CD74(-/-) macrophages. In in vivo experiments assessing the link between MIF and CD74, combined administration of MIF and CCL2 increased leukocyte adhesion in both MIF(-/-) and CD74(-/-) mice, showing that CD74 was not required for this MIF-induced response. Additionally, although leukocyte recruitment induced by administration of MIF alone was reduced in CD74(-/-) mice, consistent with a role for CD74 in leukocyte recruitment induced by MIF, MIF-treated CD74(-/-) mice displayed residual leukocyte recruitment. These data demonstrate that MIF and CD74 play previously unappreciated roles in CCL2-induced macrophage adhesion and migration, and they indicate that MIF and CD74 mediate this effect via both common and independent mechanisms.

Macrophage migration inhibitory factor regulates neutrophil chemotactic responses in inflammatory arthritis in mice.

OBJECTIVE: Macrophage migration inhibitory factor (MIF) facilitates multiple aspects of inflammatory arthritis, the pathogenesis of which has been significantly linked to the activity of neutrophils. The effects of MIF on neutrophil recruitment are unknown. This study was undertaken to investigate the contribution of MIF to the regulation of neutrophil chemotactic responses. METHODS: K/BxN serum-transfer arthritis was induced in wild-type (WT), MIF(-/-) , and monocyte chemotactic protein 1 (MCP-1; CCL2)-deficient mice as well as in WT mice treated with monoclonal antibodies to cytokine-induced neutrophil chemoattractant (anti-KC). Leukocyte trafficking in vivo was examined using intravital microscopy, and neutrophil function in vitro was examined using migration chambers and assessment of MAP kinase activation. RESULTS: K/BxN serum-transfer arthritis was markedly attenuated in MIF(-/-) mice, with reductions in the clinical and histologic severity of arthritis and the synovial expression of KC and interleukin-1. Arthritis was also reduced by anti-KC antibody treatment, but not in MCP-1-deficient mice. In vivo, neutrophil recruitment responses to KC were reduced in MIF(-/-) mice. Similarly, MIF(-/-) mouse neutrophils exhibited reduced chemotactic responses to KC in vitro, despite displaying unaltered chemokine receptor expression. Reduced chemotactic responses of MIF(-/-) mouse neutrophils were associated with reduced phosphorylation of p38 and ERK MAP kinases. CONCLUSION: These findings suggest that MIF promotes neutrophil trafficking in inflammatory arthritis via facilitation of chemokine-induced migratory responses and MAP kinase activation. Therapeutic MIF inhibition could limit synovial neutrophil recruitment.

Antimyeloperoxidase antibodies rapidly induce alpha-4-integrin-dependent glomerular neutrophil adhesion.

Patients with antineutrophil cytoplasmic antibodies (ANCAs) frequently develop severe vasculitis and glomerulonephritis. Although ANCAs, particularly antimyeloperoxidase (anti-MPO), have been shown to promote leukocyte adhesion in postcapillary venules, their ability to promote adhesion in the glomerular vasculature is less clear. We used intravital microscopy to examine glomerular leukocyte adhesion induced by anti-MPO. In mice pretreated with LPS, 50 microg anti-MPO induced LFA-1-dependent adhesion in glomeruli. In concert with this finding, in mice pretreated with LPS, more than 80% of circulating neutrophils bound anti-MPO within 5 minutes of intravenous administration. However, even in the absence of LPS, more than 40% of circulating neutrophils bound anti-MPO in vivo, a response not seen in MPO(-/-) mice. In addition, a higher dose of anti-MPO (200 microg) induced robust glomerular leukocyte adhesion in the absence of LPS. The latter response was beta2-integrin independent, instead requiring the alpha4-integrin, which was up-regulated on neutrophils in response to anti-MPO. These data indicate that anti-MPO antibodies bind to circulating neutrophils, and can induce glomerular leukocyte adhesion via multiple pathways. Lower doses induce adhesion only after an infection-related stimulus, whereas higher doses are capable of inducing responses in the absence of an additional inflammatory stimulus, via alternative adhesion mechanisms.

Molecular and cellular basis of microvascular perfusion deficits induced by Clostridium perfringens and Clostridium septicum.

Reduced tissue perfusion leading to tissue ischemia is a central component of the pathogenesis of myonecrosis caused by Clostridium perfringens. The C. perfringens alpha-toxin has been shown capable of inducing these changes, but its potential synergy with perfringolysin O (theta-toxin) is less well understood. Similarly, Clostridium septicum is a highly virulent causative agent of spontaneous gas gangrene, but its effect on the microcirculation has not been examined. Therefore, the aim of this study was to use intravital microscopy to examine the effects of C. perfringens and C. septicum on the functional microcirculation, coupled with the use of isogenic toxin mutants to elucidate the role of particular toxins in the resultant microvascular perfusion deficits. This study represents the first time this integrated approach has been used in the analysis of the pathological response to clostridial toxins. Culture supernatants from wild-type C. perfringens induced extensive cell death within 30 min, as assessed by in vivo uptake of propidium iodide. Furthermore, significant reductions in capillary perfusion were observed within 60 min. Depletion of either platelets or neutrophils reduced the alteration in perfusion, consistent with a role for these blood-borne cells in obstructing perfusion. In addition, mutation of either the alpha-toxin or perfringolysin O structural genes attenuated the reduction in perfusion, a process that was reversed by genetic complementation. C. septicum also induced a marked reduction in perfusion, with the degree of microvascular compromise correlating with the level of the C. septicum alpha-toxin. Together, these data indicate that as a result of its ability to produce alpha-toxin and perfringolysin O, C. perfringens rapidly induces irreversible cellular injury and a marked reduction in microvascular perfusion. Since C. septicum induces a similar reduction in microvascular perfusion, it is postulated that this function is central to the pathogenesis of clostridial myonecrosis, irrespective of the causative bacterium.

Independent roles of macrophage migration inhibitory factor and endogenous, but not exogenous glucocorticoids in regulating leukocyte trafficking.

OBJECTIVES: Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment and antagonizes the anti-inflammatory effects of glucocorticoids (GC). The aim of this study was to examine whether interaction between MIF and GC underlies the ability of MIF to promote leukocyte-endothelial cell (EC) interactions. METHODS: Intravital microscopy was used to assess leukocyte-EC interactions in wild-type and MIF(-/-) mice following treatment with lipopolysaccharide (LPS), the GC dexamethasone, and inhibition of endogenous GC, using the GC-receptor antagonist, RU486. RESULTS: Dexamethasone reduced LPS-induced leukocyte interactions in wild-type mice to levels similar to those observed in MIF(-/-) mice not treated with dexamethasone, whereas in MIF(-/-) mice, leukocyte interactions were not further inhibited by dexamethasone. RU486 increased LPS-induced leukocyte adhesion and emigration to a similar extent in both wild-type and MIF(-/-) mice, indicating that endogenous GC exert a similar inhibitory effect on leukocyte trafficking in wild-type and MIF(-/-) mice. Both MIF deficiency and RU486 treatment reduced VCAM-1 expression, while neither treatment modulated expression of ICAM-1 or chemokines CCL2, KC, and MIP-2. CONCLUSIONS: These results suggest that endogenous MIF and GC regulate leukocyte-EC interactions in vivo reciprocally but through predominantly independent mechanisms, and that the anti-inflammatory effect of MIF deficiency is comparable to that of exogenous GC.

Activation of the sympathetic nervous system modulates neutrophil function.

Emerging evidence has revealed that noradrenaline (NA), the main neurotransmitter of the sympathetic nervous system (SNS), regulates a variety of immune functions via binding to adrenergic receptors present on immune cells. In this study, we examined the role of NA in the regulation of neutrophil functions. Neutrophils were isolated from the bone marrow of naïve mice and treated with NA at various concentrations to assess the effect on various neutrophil functions. Additionally, we performed cremaster intravital microscopy to examine neutrophil-endothelial cell interactions following NA superfusion in vivo. In a separate group of animals, mice were subjected to an experimental model of stroke and at 4 and 24 h neutrophils were isolated for assessment on their ability to migrate toward various chemokines. Treatment of neutrophils with NA for 4 h significantly impaired neutrophil chemotaxis and induced an N2 neutrophil phenotype with reduced expression of the genes critical for cytoskeleton remodeling and inflammation. Prolonged NA administration promoted neutrophils to release myeloperoxidase and IL-6, but suppressed the production of interferon-γ and IL-10, reduced neutrophil activation and phagocytosis. Superfusion of NA over the cremaster muscle almost completely inhibited fMLP-induced neutrophil adhesion/arrest and transmigration. Furthermore, using a mouse model of stroke, a pathological condition in which SNS activation is evident, neutrophils isolated from poststroke mice showed markedly reduced chemotaxis toward all of the chemokines tested. The findings from our study indicate that neutrophil chemotaxis, activation, and phagocytosis can all be negatively regulated in an NA-dependent manner. A better understanding of the relationship between sympathetic activation and neutrophil function will be important for the development of effective antibacterial interventions.

Effector CD4+ T cells recognize intravascular antigen presented by patrolling monocytes.

Although effector CD4+ T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4+ T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4+ T cells. Following intravascular deposition of antigen in glomeruli, effector CD4+ T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII+ immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4+ T-cell-dependent glomerular inflammation. These findings indicate that MHCII+ monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4+ T cells within glomerular capillaries, leading to antigen-dependent inflammation.

Activated Renal Dendritic Cells Cross Present Intrarenal Antigens After Ischemia-Reperfusion Injury.

BACKGROUND: The healthy kidney contains an extensive population of renal mononuclear phagocytes (RMPs), with substantial phenotypic and functional diversity. However, how this diverse population is affected by ischemia-reperfusion injury (IRI), an obligate part of renal transplantation, is not yet well understood. The aim of this study was to characterize the phenotypic and functional alterations in RMPs induced by IRI. METHODS: Renal mononuclear phagocytes were studied 24 and 72 hours after 30 minutes of renal ischemia or sham operation. Kidneys were digested and the phenotypes of renal leukocyte populations were analyzed via flow cytometry. Multiphoton microscopy was used to image renal dendritic cells (DCs) in vivo using CD11c reporter mice. The capacity of renal DCs to present antigen was examined by assessment of proliferation of ovalbumin-specific T cells in rat insulin promoter-membrane-bound ovalbumin transgenic mice after sham or IRI procedures. RESULTS: Ischemia-reperfusion injury induced influx of monocytes, DCs, macrophages, and neutrophils into the kidney. Classification of RMP subpopulations based on CD11b/CD11c expression demonstrated that the RMPs that increased in response to IRI were predominantly newly recruited monocyte-derived inflammatory DCs. In vivo multiphoton imaging of CD11c-EYFP mice revealed that intrarenal DCs exhibited increased number and activity of dendrites in the postischemic period. Ischemia-reperfusion injury also promoted DC-dependent cross-presentation of renal antigens to CD8 T cells in the draining lymph node. CONCLUSIONS: In response to renal IRI, RMP populations are skewed toward those derived from inflammatory monocyte precursors. In addition, renal DCs undergo functional activation, increasing their capacity to activate antigen-specific CD8 T cells in renal draining lymph nodes.

Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus.

In contrast with many capillary beds, the glomerulus readily supports leukocyte recruitment. However, little is known regarding the actions of leukocytes following their recruitment to glomeruli. We used multiphoton confocal microscopy to examine leukocyte behavior in the glomerular microvasculature. In normal glomeruli, neutrophils and monocytes were retained in capillaries for several minutes, remaining static or migrating intravascularly. Induction of glomerular inflammation resulted in an increase in the duration of retention of static and migratory leukocytes. In response to immune complex deposition, both static and migratory neutrophils generated oxidants in inflamed glomeruli via a Mac-1-dependent mechanism. Our results describe a new paradigm for glomerular inflammation, suggesting that the major effect of acute inflammation is to increase the duration of leukocyte retention in the glomerulus. Moreover, these findings describe a previously unknown form of multicellular intravascular patrolling that involves both monocytes and neutrophils, which may underlie the susceptibility of the glomerulus to inflammation.