Neutrophil Retention Research Articles

Modulation of Neutrophil Function by a Secreted Mucinase of Escherichia coli O157∶H7

Escherichia coli O157∶H7 is a human enteric pathogen that causes hemorrhagic colitis which can progress to hemolytic uremic syndrome, a severe kidney disease with immune involvement. During infection, E. coli O157∶H7 secretes StcE, a metalloprotease that promotes the formation of attaching and effacing lesions and inhibits the complement cascade via cleavage of mucin-type glycoproteins. We found that StcE cleaved the mucin-like, immune cell-restricted glycoproteins CD43 and CD45 on the neutrophil surface and altered neutrophil function. Treatment of human neutrophils with StcE led to increased respiratory burst production and increased cell adhesion. StcE-treated neutrophils exhibited an elongated morphology with defective rear detachment and impaired migration, suggesting that removal of the anti-adhesive capability of CD43 by StcE impairs rear release. Use of zebrafish embryos to model neutrophil migration revealed that StcE induced neutrophil retention in the fin after tissue wounding, suggesting that StcE modulates neutrophil-mediated inflammation in vivo. Neutrophils are crucial innate effectors of the antibacterial immune response and can contribute to severe complications caused by infection with E. coli O157∶H7. Our data suggest that the StcE mucinase can play an immunomodulatory role by directly altering neutrophil function during infection. StcE may contribute to inflammation and tissue destruction by mediating inappropriate neutrophil adhesion and activation.

Crosstalk between CXCR4/Stromal Derived Factor-1 and VLA-4/VCAM-1 Pathways Regulates Neutrophil Retention in the Bone Marrow

Neutrophil retention in and release from the bone marrow is a critical process that remains incompletely understood. Previous work has implicated the CXCR4/stromal derived factor-1 (SDF-1) chemokine axis in the marrow retention of neutrophils, yet the adhesion pathways responsible for this retention are unknown. Because alpha(4)beta(1) integrin (VLA-4) and its ligand VCAM-1 play a central role in the interactions of hematopoietic stem cells, lymphocytes, and developing neutrophils in the marrow, we investigated whether this integrin might be involved in marrow neutrophil retention and release. In this study, we show that VLA-4 is expressed on murine marrow neutrophils and decreases with maturation, whereas blockade of this integrin leads to the release of marrow neutrophils. Marrow neutrophils adhere via VLA-4 to VCAM-1, which is expressed on marrow endothelium and stroma, and inhibition of VCAM-1 causes release of marrow neutrophils. Furthermore, SDF-1 (CXCL12) signaling through neutrophil CXCR4 augments VLA-4 adhesion to VCAM-1 in vitro, an effect that is blocked by preincubation with pertussis toxin. In vivo blockade of both CXCR4 and alpha(4) causes synergistic release of marrow neutrophils, showing that cross-talk between CXCR4 and VLA-4 modulates marrow retention of these cells. Taken together, these results indicate that the VLA-4/VCAM adhesion pathway is critical in the retention and maturation-controlled release of neutrophils from the marrow, while providing an important link between the CXCR4/SDF-1 signaling axis and the adhesion events that govern this process.

Therapeutic Targeting of IL-6 Trans Signaling Counteracts STAT3 Control of Experimental Inflammatory Arthritis

Cytokine control of the synovial infiltrate is a central process in the development of inflammatory arthritis. In this study, we combine genetic approaches and intervention strategies to describe a fundamental requirement for IL-6-mediated STAT3 signaling in orchestrating the inflammatory infiltrate in monoarticular and systemic models of experimental arthritis. STAT3 activation via the common gp130 signal-transducing receptor for all IL-6-related cytokines led to increased retention of neutrophils and T cells within the inflamed synovium, which included STAT3-regulated IL-17A-secreting T cells. Control of leukocyte infiltration was reliant upon IL-6 signaling via its soluble receptor (termed IL-6 trans signaling), as evidenced by selective blockade of this alternative IL-6 signaling pathway using an engineered variant of soluble gp130 (sgp130Fc). This therapeutic intervention led to substantial clinical improvement in mice with emerging or established incidence of systemic arthritis. These data illustrate that IL-6 control of STAT3 is critical for regulating the synovial infiltrate in inflammatory arthritis, and suggest that selective inhibition of IL-6 trans signaling may provide a more refined intervention strategy for blocking IL-6-driven proarthritic activities.

Rapid and recurrent neutrophil mobilization regulated by T134, a CXCR4 peptide antagonist

The CXCR4/stromal cell-derived factor-1 (SDF-1) axis plays important roles in development, leukocyte trafficking, HIV infection, and tumorigenesis. Its critical function in bone marrow stem cell and hematopoietic progenitor cell retention, homing and release has been well-characterized by genetic and pharmacological analyses. However, its role in neutrophil retention and release is still poorly understood. In this study, we demonstrated that T134, a peptide antagonist of human CXCR4, is also a potent antagonist of mouse CXCR4. Treatment of C57BL/6 mice with T134 resulted in a rapid and time-dependent increase of white blood cells (WBC) and neutrophils, as well as hematopoietic stem and progenitor cells in peripheral blood. Interestingly, recurrent WBC and neutrophil mobilization was achieved by repeated T134 treatment, and the T134-mediated increase and subsequent retreat of WBC and neutrophils correlated with T134 activity in the peripheral blood. Kinetic analysis revealed that T134 binding to CXCR4 did not induce any significant cell-surface receptor downregulation, indicating that T134-induced WBC and neutrophil mobilization is likely due to direct blockage of the CXCR4/SDF-1 interaction. The results from this study support an important role of CXCR4/SDF-1 axis in neutrophil retention and release in the marrow.

Surface Expression of Neutrophil CXCR4 is Down-modulated by Bacterial Endotoxin

The chemokine receptor CXCR4 and its unique ligand, stromal-derived factor 1 (SDF-1), play critical roles in the retention of hematopoietic cells within bone marrow and in their mobilization into the circulation. Surface CXCR4 down-regulation in hematopoietic cells is associated with a loss of retention of the cells in bone marrow. Lipopolysaccharide (LPS), commonly referred to as endotoxin, induces neutrophilia in vivo, but the mechanism of mobilization related to neutrophilia has not been fully clarified. We show that LPS reduces CXCR4 surface expression in a dose- and time-dependent manner in neutrophils and monocytes, but not in lymphocytes. Polymyxin B neutralization of LPS in culture supernatants still induced this down-modulation, and LPS-stimulated neutrophils released interferon gamma and interleukin 1beta. These results provide evidence that CXCR4 down-regulation can be attributed to soluble factors released by neutrophils upon LPS treatment. Moreover, LPS treatment increased CXCR4 messenger RNA in neutrophils, suggesting that the down-regulation of surface CXCR4 is caused by a posttranslational mechanism, and the chemotactic migration of neutrophils in response to SDF-1 was reduced by LPS pretreatment. Thus, the present study has shown that by down-regulating neutrophil CXCR4 expression and attenuating neutrophil responsiveness to SDF-1, LPS can mobilize neutrophils from bone marrow to the peripheral blood through reducing neutrophil retention in bone marrow.

Phosphoinositide 3-kinase signalling and FoxO transcription factors in rheumatoid arthritis

Although the mechanisms leading to the induction of RA (rheumatoid arthritis) are poorly understood, improper activation, proliferation, survival and retention of neutrophils, macrophages, lymphocytes and other leucocytes contribute to perpetuation of inflammation and eventual joint destruction through activation of stromal fibroblast-like synoviocytes. Fundamental studies in developmental biology, cellular biology and immunology have established critical roles for PI3K (phosphoinositide 3-kinase) signal transduction pathways in cellular chemotactic responses, proliferation, apoptosis and survival. Despite profound alteration of these cellular processes in RA, involvement of PI3K signalling pathways in this chronic inflammatory disease, and their assessment as potential therapeutic targets, has until recently received scant attention. This review highlights recent advances in our understanding of PI3K signalling pathways, in particular regulation of FoxO (forkhead box O) transcription factors, and their relevance to RA.

G-CSF down-regulation of CXCR4 expression identified as a mechanism for mobilization of myeloid cells

AbstractCXCR4 receptor expression is required for the retention of granulocyte precursors and mature neutrophils within the bone marrow, and disruption of the SDF-1/CXCR4 axis in the bone marrow results in the mobilization of myeloid lineage cells to the peripheral circulation. We report that G-CSF down-regulates CXCR4 expression in bone marrow–derived murine and human myeloid lineage cells. When exposed to G-CSF, murine Gr1+ bone marrow myeloid cells display a time-dependent reduction of cell-surface CXCR4 and respond poorly to SDF-1 in attachment and migration assays. Bone marrow–derived cells of nonmyeloid lineage display no change in surface CXCR4 expression upon exposure to G-CSF. Compared with controls, mice treated with G-CSF for mobilization of hematopoietic progenitor cells display reduced levels of CXCR4 selectively in bone marrow Gr1+ myeloid cells. Since bone marrow myeloid cells express G-CSF receptors and G-CSF rapidly reduces CXCR4 expression in purified Gr1+ cells populations, these results provide evidence that G-CSF acts directly on myeloid lineage cells to reduce CXCR4 expression. By down-regulating CXCR4 expression in bone marrow myeloid cells and attenuating their responsiveness to SDF-1, G-CSF promotes their mobilization from the bone marrow to the peripheral blood.

The WHIM syndrome shows a peculiar dysgranulopoiesis: myelokathexis

A 17-year-old boy had been born at term after an uneventful pregnancy to non-consanguineous parents. His siblings and parents were well. He had experienced numerous upper respiratory tract infections since birth, which had caused pulmonary atelectasia requiring surgical removal of the affected lobe. He had suffered recurrent herpetic peribuccal infection. Warts had appeared on his hands at 6 years of age and extended gradually to the elbows, arms, legs, knees and feet. Pancytopenia was first documented at the age of 1 month. White blood cell counts varied from 1·9 to 3·9 × 109/l with neutrophil counts of 0·9–2·8 × 109/l. There was no cyclic pattern of neutropenia. His haemoglobin concentration ranged between 10 and 12 g/dl and the platelet count was 47–127 × 109/l. Blood smears showed abnormal neutrophils with condensed nuclei connected by long, stringy filaments. The bone marrow was hypercellular with an increased proportion of mature myeloid cells (left). Many neutrophils had bisegmented nuclei with dense pyknotic lobes connected by long filaments leading to bizarre forms, such as ‘eyeglasses’ or ‘clover leaf’ (right). Occasionally, these filaments were twisted around each other. Some eosinophils showed cytoplasmic vacuolation. No significant morphological abnormalities were observed in the other cell lineages. Folic acid and vitamin B12 levels were normal. Immunological investigation showed severe combined immunodeficiency. Both B- and T-cell subpopulations were reduced, particularly CD4+ T cells. T-cell responses to vaccine antigens were moderately affected. Global hypogammaglobulinaemia was observed (IgG < 6 g/l; normal levels of IgG2 and IgG3). No antibodies to leucocytes or platelets were detected and karyotype analysis was normal. Monthly treatment with gammaglobulins and filgrastim was initiated when the child was 7 years of age, producing a subsequent decrease in the rate of infections and an increased neutrophil count (>1 × 109/l). The patient presented the full spectrum of findings described in the WHIM syndrome, a rare immunodeficiency disorder characterised by warts, hypogammaglobulinaemia, infections and myelokathexis. Myelokathexis is characterised by a prolonged retention of neutrophils in the bone marrow compartment leading to neutropenia, despite hyperplasia of bone marrow myeloid cells, and degenerative changes in mature neutrophils. The observation of these morphologic features was strongly suggestive of myelokathexis and is helpful in the diagnosis of WHIM syndrome. This syndrome is caused by the mutation of a chemokine receptor gene, leading to production of the mutant CXCR4 protein that causes abnormal aptoptosis and migratory function, thought to be related to the cause of chronic leucopenia.

Mutant CXCR4 Identified in Neutropenic Patients with Myelokathexis Impairs Survival of Human Myeloid Cells.

Myelokathexis (WHIM syndrome) is a very rare hematopoietic congenital disorder that is characterized by extremely low level of circulating neutrophils in peripheral blood. It is inherited as an autosomal dominant disease and is diagnosed in early childhood. These patients may have hypogammaglobulinemia and suffer from recurrent infections associated with warts. The hallmark of myelokathexis is a hyperplastic bone marrow and hypersegmented neutrophils with nuclear lobs connected with thin filaments. Myelokathexis is due to a characteristic retention of mature neutrophils in bone marrow, which are not being released to peripheral circulation. We and others reported abnormal cell survival characteristics and impaired bcl-x expression in bone marrow myeloid cells of myelokathexis patients that was partially restored by G-CSF treatment. Recently, it has also been reported that heterozygous truncation mutations in the carboxyterminal domain of the CXCR4 gene, a sole receptor for SDF-1 chemokine, were observed in most, but not all of the families with WHIM syndrome. Subsequently, an impaired receptor internalization and increased chemotaxis towards SDF-1 have been observed in cells expressing truncated CXCR4. Nevertheless, the mechanism of mutant CXCR4 induced myelokathexis remains largely unknown.We performed mutational analysis of the CXCR4 gene in 3 unrelated families with myelokathexis and identified a previously reported R334ter truncation mutation in exon 2 in two of the families. In addition, two silent polymorphisms have been identified in exon 2 of the CXCR4 gene in one of these patients. The third family with afflicted mother and son had a new mutation in the CXCR4 carboxyterminal domain, which resulted in deletion of the last 16 amino acids and subsequent frame shift. None of these mutations were observed in healthy volunteers examined. Since the morphological examination by electron microscopy and flow cytometry analysis of bone marrow cells from some of these patients revealed characteristic apoptotic features, we examined the effect of mutant CXCR4 gene expression on survival of human promyelocytic HL-60 cells. Preliminary data demonstrated that human promyelocytic cells transfected with truncated CXCR4 exhibited impaired cell survival characteristics compared with control HL-60 cells transfected with intact CXCR4. The truncated, but not wild type CXCR4 also increased apoptosis in HL-60 cells induced to differentiate along the granulocytic pathway as determined by flow cytometry of annexin V labeled cells. Thus, these data link together the abnormal survival of proliferating and differentiating myeloid cells in WHIM syndrome with mutant CXCR4 expression. Current studies are focused on elucidation of specific signaling pathways mediating mutant CXCR4-triggered myelokathexis.

Role of Adenosine Receptors in Volatile Anesthetic Preconditioning against Neutrophil-induced Contractile Dysfunction in Isolated Rat Hearts

The authors tested the hypothesis that adenosine receptors in polymorphonuclear neutrophils and the heart mediate the preconditioning effects of volatile anesthetics against neutrophil-induced contractile dysfunction. Studies were conducted in buffer-perfused and paced isolated rat hearts. Left ventricular developed pressure served as index of contractility. Neutrophils and platelet-activating factor were added to perfusate for 10 min followed by 30 min of recovery. The effect of selective pretreatment of the neutrophils and the hearts with 1.0 minimum alveolar concentration isoflurane or sevoflurane on the neutrophil-induced contractile dysfunction was assessed. Studies were performed in the absence and presence of the nonselective adenosine receptor antagonist 8-phenyltheophylline (10 microM). Neutrophil retention was determined from difference between those administered and collected in coronary effluent and from myeloperoxidase concentration in myocardial samples. Superoxide production of neutrophils was measured by spectrophotometry. Under control conditions (no anesthetic pretreatment), activated neutrophils caused marked and persistent reductions in left ventricular developed pressure, associated with increases in neutrophil retention and myeloperoxidase activity. Pretreatment of the neutrophils or the heart with either isoflurane or sevoflurane abolished these effects. Pretreatment of the neutrophils also reduced the platelet-activating factor-induced increase in superoxide production by 29 and 33%, respectively. 8-Phenyltheophylline blunted the effects of anesthetic pretreatment of the neutrophils, whereas it did not alter the effects of anesthetic pretreatment of the heart. An activation of adenosine receptors in neutrophils, but not in the heart, plays a role in the preconditioning effects of volatile anesthetics against neutrophil-induced contractile dysfunction.

TNF-α promotes a stop signal that inhibits neutrophil polarization and migration via a p38 MAPK pathway

Neutrophils are a major component of the inflammatory response in patients with asthma and other inflammatory conditions. Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), are increased in the airway of patients with severe asthma and have been implicated in the recruitment of neutrophils into areas of inflammation. Here, we show that TNF-alpha induces a stop signal that promotes firm neutrophil adhesion and inhibits neutrophil polarization and chemotaxis to chemoattractants including interleukin-8 and C5a. TNF-alpha treatment of neutrophils plated on a fibrinogen-coated surface promotes firm neutrophil adhesion and the formation of vinculin-containing focal complexes. TNF-alpha induces a more than tenfold increase in p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase phosphorylation. Inhibition of p38 MAPK in neutrophils treated with TNF-alpha causes neutrophil polarization and motility. These findings suggest that TNF-alpha initiates a stop signal through a p38 MAPK pathway, which may promote the retention of neutrophils in inflammatory sites. Together, our data suggest that inhibition of p38 MAPK may be an attractive target to limit inflammatory responses that are mediated by TNF-alpha.

The CXC chemokine MIP-2 stimulates neutrophil mobilization from the rat bone marrow in a CD49d-dependent manner

AbstractThe acute release of neutrophils from the bone marrow is a critical step in their trafficking to sites of inflammation. This process is stimulated by systemically acting inflammatory mediators, such as the CXC chemokines. In this study we have used a novel in situ perfusion system of the rat femoral bone marrow to directly investigate the role of specific adhesion molecules in chemokine-stimulated neutrophil mobilization. We show here that neutrophils mobilized in response to rat macrophage inflammatory protein-2 (MIP-2) shed l-selectin and expressed significantly higher levels of CD11b and CD49d. However, inhibition of l-selectin sheddase activity with KD-IX-73-4 had no effect on the number of neutrophils mobilized in response to rat MIP-2. Blockade of CD18, using a neutralizing monoclonal antibody (mAb), did not inhibit neutrophil mobilization but unexpectedly increased the rate and number of neutrophils released from the bone marrow in response to chemokine, suggesting that CD18 could play a role in neutrophil retention within the bone marrow. Blockade of CD49d using either a selective mAb or a specific antagonist resulted in a dramatic inhibition (> 75%) of the chemokine-stimulated neutrophil mobilization from the bone marrow. These data reveal contrasting roles for CD18 and CD49d in the retention and release of neutrophils from the bone marrow.

Inhibition of c-Jun N-Terminal Kinase Limits Lipopolysaccharide-induced Pulmonary Neutrophil Influx

The influx of neutrophils into the lung is a sentinel event in LPS-induced acute lung inflammation. Previous studies have shown that systemic inhibition of p38 decreases LPS-induced neutrophil influx into the alveolar space but has no effect on pulmonary parenchymal neutrophil accumulation or on microvascular leak, indicating other pathways are important in LPS-induced acute lung inflammation. This study examined the role of c-Jun N-terminal kinase in LPS-induced acute lung inflammation. Systemic inhibition of c-Jun N-terminal kinase, with the specific c-Jun N-terminal kinase inhibitor SP600125, decreased the LPS-induced accumulation of neutrophils into the lung parenchyma and alveolar space. In addition, increases in microvascular leak after LPS exposure were diminished by c-Jun N-terminal kinase inhibition. To determine mechanisms by which systemic c-Jun N-terminal kinase inhibition decreased pulmonary neutrophil influx, LPS and tumor necrosis factor alpha (TNF-alpha-)-induced neutrophil actin assembly and retention were examined. Neutrophil actin assembly was decreased after LPS and TNF-alpha stimulation with SP600125 pretreatment, as well as LPS-induced neutrophil retention. Finally, c-Jun N-terminal kinase inhibition decreased Cdc42 activation after LPS or TNF-alpha stimulation, thereby providing one mechanism by which c-Jun N-terminal kinase inhibition decreased actin assembly, and thereby pulmonary neutrophil accumulation.

FK419, a Novel Nonpeptide GPIIb/IIIa Antagonist, Restores Microvascular Patency and Improves Outcome in the Guinea-Pig Middle Cerebral Artery Thrombotic Occlusion Model: Comparison with Tirofiban

The antithrombotic efficacy of FK419, a novel nonpeptide platelet glycoprotein IIb/IIIa antagonist, was compared with tirofiban in guinea-pigs. FK419 and tirofiban similarly inhibited platelet aggregation in vitro (IC50 values: 0.43+/-0.076 and 0.41+/-0.053 micromol/L) and dispersed aggregated platelets (EC50 values: 2.3+/-0.88 and 2.0+/-0.81 micromol/L). FK419 inhibited retention of platelets and neutrophils in a collagen-coated bead column with greater potency than tirofiban (IC50 values of 0.90+/-0.133 and 2.4+/-0.21 micromol/L for platelet retention and 0.32+/-0.078 and 0.57+/-0.180 micromol/L for neutrophil retention). When FK419 or tirofiban were administered after photochemically induced middle cerebral artery (MCA) occlusion in guinea-pigs, they dose-dependently improved MCA patency. FK419 reduced neurological deficits and ischemic brain damage in a dose-dependent fashion, whereas tirofiban did not. Reduced regional cerebral blood flow in the striatum gradually returned to the preoccluded level with FK419 treatment; however, no restoration was observed with tirofiban even though the MCA was recanalized. These results indicate that FK419 ameliorates ischemic brain damage by not only lysing the obstructive thrombus in MCA but also preventing or restoring microcirculation deficits after occlusion/reperfusion, suggesting that FK419 would be an attractive intervention for the treatment of ischemic stroke patients.

Isoflurane and sevoflurane precondition against neutrophil-induced contractile dysfunction in isolated rat hearts.

The authors tested the hypothesis that pretreatment with isoflurane or sevoflurane can protect the heart against neutrophil-induced contractile dysfunction. Studies were conducted in buffer-perfused and paced isolated rat hearts. Left ventricular developed pressure served as an index of contractility. Pretreatment consisted of administration of 1.0 minimum alveolar concentration isoflurane or sevoflurane for 15 min followed by a 10-min washout and was performed in the absence and presence of the adenosine triphosphate-sensitive potassium channel inhibitor glibenclamide (10 microM). Polymorphonuclear neutrophils and platelet-activating factor were then added to the perfusate for 10 min, followed by 30 min of recovery. Neutrophil retention was assessed from the difference between those administered and collected in coronary effluent and measurements of myeloperoxidase in myocardial samples. Isolated hearts were also used to assess the effect of volatile anesthetic pretreatment on cardiac dysfunction caused by enzymatically generated superoxide. In additional studies, the authors evaluated the effect of volatile anesthetic pretreatment on the adherence of neutrophils to isolated rat aortic segments. Platelet-activating factor-stimulated neutrophils caused marked and persistent reductions (> 50%) in left ventricular developed pressure. Pretreatment with either isoflurane or sevoflurane abolished these effects, as well as the associated increases in neutrophil retention. Glibenclamide did not alter these actions of the anesthetics. Pretreatment with either volatile anesthetic attenuated the reductions in left ventricular developed pressure caused by exogenous superoxide and abolished the increases in neutrophil adherence in the aortic segments. Isoflurane and sevoflurane preconditioned the heart against neutrophil-induced contractile dysfunction. This action was associated with an inhibition to neutrophil adherence and likely involved an increased resistance of the myocardium to oxidant-induced injury; the adenosine triphosphate-sensitive potassium channels played no apparent role.

Chemokines Acting via CXCR2 and CXCR4 Control the Release of Neutrophils from the Bone Marrow and Their Return following Senescence

In this study we provide evidence that the SDF-1α/CXCR4 chemokine axis is involved in both the retention of neutrophils within the bone marrow and the homing of senescent neutrophils back to the bone marrow. We show that the functional responses of freshly isolated human and murine neutrophils to CXCR2 chemokines are significantly attenuated by SDF-1α, acting via CXCR4. As a consequence, the mobilization of neutrophils from the bone marrow in vivo by the CXCR2-chemokine, KC, was dramatically enhanced by blocking the effects of endogenous SDF-1α using a specific CXCR4 antagonist. As neutrophils age, they upregulate expression of CXCR4 and acquire the ability to migrate toward SDF-1α. We show here that these senescent CXCR4 high neutrophils preferentially home to the bone marrow in vivo in a CXCR4-dependent manner, suggesting a previously undefined mechanism for the clearance of senescent neutrophils from the circulation.

Effect of antibodies to intercellular adhesion molecule type 1 on the protection of distant organs during reperfusion syndrome in rats.

We investigated kidney and lung alterations caused by intercellular adhesion molecule type 1 (ICAM-1) blockade after ischemia and reperfusion of hind limb skeletal muscles. Rats were submitted to ligature of the infrarenal aorta for 6 h. The animals were randomized into three groups of 6 rats each: group I, sacrificed after ischemia; group II, reperfusion for 24 h, and group III, reperfusion for 24 h after receiving monoclonal anti-ICAM-1 antibodies. At the end of the experiment, blood samples were collected for creatinine, lactate dehydrogenase, creatine phosphokinase, potassium, pH and leukocyte counts. Samples were taken from the muscles of the hind limbs and from the kidneys and lungs for histological analysis and measurement of the neutrophil infiltrate by myeloperoxidase staining. The groups did not differ significantly with regard to the laboratory tests. There were no major histological alterations in the kidneys. An intense neutrophil infiltrate in the lungs, similar in all groups, was detected. Myeloperoxidase determination showed that after reperfusion there was significantly less retention of polymorphonuclear neutrophils in the muscles (352 +/- 70 vs 1451 +/- 235 x 10(2) neutrophils/mg; P<0.01) and in the kidneys (526 +/- 89 vs 852 +/- 73 10(2) neutrophils/mg; P<0.01) of the animals that received anti-ICAM-1 before perfusion compared to the group that did not. The use of anti-ICAM-1 antibodies in this experimental model minimized neutrophil influx, thus reducing the inflammatory process, in the muscles and kidneys after ischemia and reperfusion of the hind limbs.

Selectin-mediated rolling of neutrophils is essential for their activation and retention in the reperfused coronary system.

Neutrophil adhesion to coronary endothelium is a key event for cardiac reperfusion injury. Adhesion is proposed to be a multi-step event, consisting of selectin-mediated rolling, chemotactic activation, and subsequent integrin-mediated firm attachment. However, it is not clear whether this sequence also occurs in the coronary circulation with its unique hemodynamic properties (turbulent flow, flow reversal). We have studied neutrophil adhesion in the coronary system of isolated perfused guinea pig hearts under basal and reperfusion conditions (15 min global ischemia). Adhesion was manipulated by an anti-CD18 antibody (blocking firm adhesion) and fucoidin (reducing rolling). Neutrophil behavior during coronary passage was assessed by measurement of CD11b expression, forward scatter (FSC, indicating polarization), and sideward scatter (SSC, measure for granularity) via flow cytometry. Adhesion rose from 21 % (basal) to 35 % after ischemia. Anti-CD18 decreased adhesion to 11 % and 14 %, respectively; fucoidin altered only the postischemic increase (23 %). CD11b was unchanged by passage through the non-ischemic coronaries, but rose postischemically (139 % increase). CD18 blockade did not reduce the postischemic rise of CD11b, while fucoidin was inhibitory (24 % increase). FSC did not differ between controls and ischemic hearts in any group, while SSC decreased most in postischemic hearts after CD18 blockade. Blockade of rolling and of firm attachment both reduce neutrophil retention, while only inhibition of rolling reduces intracoronary activation. Thus, rolling seems to be mandatory for endothelial-leukocyte communication in the coronary system.

Polynitroxyl-albumin (PNA) plus tempol attenuate lung capillary leak elicited by prolonged intestinal ischemia and reperfusion

Stable nitroxyl radicals (nitroxides) are potential antioxidant drugs, and we have previously reported that linking nitroxide to biological macromolecules can improve therapeutic activity in at least two ways. First, polynitroxylated compounds such as polynitroxyl human serum albumin (PNA) are a novel class of high molecular weight, extracellular antioxidants. Second, compounds such as PNA can prolong the half-life of free (unbound, low molecular weight) nitroxides such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol) in vivo. Unlike PNA, Tempol can readily access the intracellular compartment. Thus PNA can act alone in the extracellular compartment, or in concert with Tempol, to provide additional antioxidant protection within cells. In this study, we compared the abilities of PNA, Tempol, and the combination of PNA + Tempol to prevent lung microvascular injury secondary to prolonged gut ischemia (I, 120 min) and reperfusion (R, 20 min) in the rat. Pulmonary capillary filtration coefficient (K f,c) and lung neutrophil retention (tissue myeloperoxidase activity, MPO) were measured in normal, isolated rat lungs perfused with blood harvested from I/R rats. Blood donor rats were treated with drug during ischemia. Gut I/R resulted in a marked increase in pulmonary capillary coefficient and lung MPO. PNA + Tempol, but not PNA alone or Tempol alone, at the doses used, prevented the development of lung leak. None of the treatments had an effect on lung neutrophil retention. Anti-inflammatory therapeutic activity appeared to correlate with blood Tempol level: in the presence of PNA, blood Tempol levels were maintained in the 50–100 μM range vs. essentially undetectable levels shortly after Tempol was administered alone. In this model of lung injury secondary to prolonged gut I/R, lung capillary leak was prevented when the membrane-permeable compound Tempol was maintained in its active, free radical state by PNA.

Signaling Functions of L-Selectin in Neutrophils: Alterations in the Cytoskeleton and Colocalization with CD18

Ligation and clustering of L-selectin by Ab ("cross-linking") or physiologic ligands results in activation of diverse responses that favor enhanced microvascular sequestration and emigration of neutrophils. The earliest responses include a rise in intracellular calcium, enhanced tyrosine phosphorylation, and activation of extracellular signal-regulated kinases. Additionally, cross-linking of L-selectin induces sustained shape change and activation of beta2 integrins, leading to neutrophil arrest under conditions of shear flow. In this report, we examined several possible mechanisms whereby transmembrane signals from L-selectin might contribute to an increase in the microvascular retention of neutrophils and enhanced efficiency of emigration. In human peripheral blood neutrophils, cross-linking of L-selectin induced alterations in cellular biophysical properties, including a decrease in cell deformability associated with F-actin assembly and redistribution, as well as enhanced adhesion of microspheres bound to beta2 integrins. L-selectin and the beta2 integrin became spatially colocalized as determined by confocal immunofluorescence microscopy and fluorescence resonance energy transfer. We conclude that intracellular signals from L-selectin may enhance the microvascular sequestration of neutrophils at sites of inflammation through a combination of cytoskeletal alterations leading to cell stiffening and an increase in adhesiveness mediated through alterations in beta2 integrins.