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Mice deficient in CD18, which lack all four CD11 integrins, have leukocytosis and increased susceptibility to bacterial infection. To determine the effect of deficiencies in LFA-1 (CD11a/CD18) or Mac-1 (CD11b/CD18) on host defense against systemic bacterial infection, knockout mice were inoculated i.p. with Streptococcus pneumoniae. Increased mortality occurred in both LFA-1(-/-) (15 of 17 vs 13 of 35 in wild type (WT), p < 0.01) and Mac-1(-/-) (17 of 34 vs 6 of 25, p < 0.01) mice. All deaths in LFA-1(-/-) mice occurred after 72 h, whereas most deaths in Mac-1(-/-) mice occurred within 24-48 h. At 24 h, 21 of 27 Mac-1(-/-) mice were bacteremic, vs 15 of 25 WT (p = 0.05); no difference was observed between LFA-1(-/-) and WT. Increased bacteria were recovered from Mac-1(-/-) spleens at 2 h (p = 0.03) and 6 h (p = 0.002) and from livers (p = 0.001) by 6 h. No difference was observed at 2 h in LFA-1(-/-) mice, but by 6 h increased bacteria were recovered from spleens (p = 0.008) and livers (p = 0.04). Baseline and peak leukocyte counts were similar between Mac-1(-/-) and WT, but elevated in LFA-1(-/-). At 8 h, peritoneal neutrophils were increased in Mac-1(-/-), but not significantly different in LFA-1(-/-). Histopathologically, at 24 h Mac-1(-/-) animals had bacteremia and lymphoid depletion, consistent with sepsis. LFA-1(-/-) mice had increased incidence of otitis media and meningitis/encephalitis vs WT at 72 and 96 h. Both Mac-1 and LFA-1 play important but distinct roles in host defense to S. pneumoniae.

Reperfusion of the ischemic myocardium is associated with a dramatic inflammatory response leading to TNF-alpha release, IL-6 induction, and subsequent neutrophil-mediated cytotoxic injury. Because inflammation is also an important factor in cardiac repair, we hypothesized the presence of components of the inflammatory reaction with a possible role in suppressing acute injury. Thus, we investigated the role of IL-10, an anti-inflammatory cytokine capable of modulating extracellular matrix biosynthesis, following an experimental canine myocardial infarction. Using our canine model of myocardial ischemia and reperfusion, we demonstrated significant up-regulation of IL-10 mRNA and protein in the ischemic and reperfused myocardium. IL-10 expression was first detected at 5 h and peaked following 96-120 h of reperfusion. In contrast, IL-4 and IL-13, also associated with suppression of acute inflammation and macrophage deactivation, were not expressed. In the ischemic canine heart, CD5-positive lymphocytes were the predominant source of IL-10 in the myocardial infarct. In the absence of reperfusion, no significant induction of IL-10 mRNA was noted. In addition, IL-12, a Th1-related cytokine associated with macrophage activation, was not detected in the ischemic myocardium. In vitro experiments demonstrated late postischemic cardiac-lymph-induced tissue inhibitor of metalloproteinases (TIMP)-1 mRNA expression in isolated canine mononuclear cells. This effect was inhibited when the incubation contained a neutralizing Ab to IL-10. Our findings suggest that lymphocytes infiltrating the ischemic and reperfused myocardium express IL-10 and may have a significant role in healing by modulating mononuclear cell phenotype and inducing TIMP-1 expression.

On inflamed endothelium selectins support neutrophil capture and rolling that leads to firm adhesion through the activation and binding of beta 2 integrin. The primary mechanism of cell activation involves ligation of chemotactic agonists presented on the endothelium. We have pursued a second mechanism involving signal transduction through binding of selectins while neutrophils tether in shear flow. We assessed whether neutrophil rolling on E-selectin led to cell activation and arrest via beta 2integrins. Neutrophils were introduced into a parallel plate flow chamber having as a substrate an L cell monolayer coexpressing E-selectin and ICAM-1 (E/I). At shears >/=0.1 dyne/cm2, neutrophils rolled on the E/I. A step increase to 4.0 dynes/cm2 revealed that approximately 60% of the interacting cells remained firmly adherent, as compared with approximately 10% on L cells expressing E-selectin or ICAM-1 alone. Cell arrest was dependent on application of shear and activation of Mac-1 and LFA-1 to bind ICAM-1. Firm adhesion was inhibited by blocking E-selectin, L-selectin, or PSGL-1 with Abs and by inhibitors to the mitogen-activated protein kinases. A chimeric soluble E-selectin-IgG molecule specifically bound sialylated ligands on neutrophils and activated adhesion that was also inhibited by blocking the mitogen-activated protein kinases. We conclude that neutrophils rolling on E-selectin undergo signal transduction leading to activation of cell arrest through beta 2 integrins binding to ICAM-1.

We examined the relative contributions of LFA-1, Mac-1, and ICAM-3 to homotypic neutrophil adhesion over the time course of formyl peptide stimulation at shear rates ranging from 100 to 800 s-1. Isolated human neutrophils were sheared in a cone-plate viscometer and the kinetics of aggregate formation was measured by flow cytometry. The efficiency of cell adhesion was computed by fitting the aggregate formation rates with a model based on two-body collision theory. Neutrophil homotypic adhesion kinetics varied with shear rate and was most efficient at 800 s-1, where approximately 40% of the collisions resulted in adhesion. A panel of blocking Abs to LFA-1, Mac-1, and ICAM-3 was added to assess the relative contributions of these molecules. We report that 1) LFA-1 binds ICAM-3 as its primary ligand supporting homotypic adhesion, although the possibility of other ligands was also detected. 2) Mac-1 binding to an unidentified ligand supports homotypic adhesion with an efficiency comparable to LFA-1 at low shear rates of approximately 100 s-1. Above 300 s-1, however, Mac-1 and not LFA-1 were the predominant molecules supporting cell adhesion. This is in contrast to neutrophil adhesion to ICAM-1-transfected cells, where LFA-1 binds with a higher avidity than Mac-1 to ICAM-1. 3) Following stimulation, the capacity of LFA-1 to support aggregate formation decreases with time at a rate approximately 3-fold faster than that of Mac-1. The results suggest that the relative contributions of beta2 integrins and ICAM-3 to neutrophil adhesion is regulated by the magnitude of fluid shear and time of stimulus over a range of blood flow conditions typical of the venular microcirculation.

Numerous studies have shown that polymorphonuclear neutrophils (PMNs) infiltrate the myocardium immediately after reperfusion of infarcted tissue. Studies with mAbs in vivo and cellular studies in vitro suggest that PMN-induced injury of the cardiac myocyte involve Mac-1 adhesion to myocyte ICAM-1. In this study we demonstrate that PMNs that have infiltrated the ischemic area begin to lose Mac-1 within the first 3 h. By the fifth hour of reperfusion, minimal CD11b staining is seen on PMNs using immunostaining, whereas CD11a remained unchanged. Immunoreactivity of postreperfusion cardiac lymph with R15.7 (anti-CD18) or MY904 (anti-CD11b) was positive in all animals but not for CD11a (R7.1), indicating a specific loss of Mac-1. Immunoprecipitation with either R15.7 or MY904 resulted in identical peptides (a doublet at 190 kDa and a band at 80 kDa), suggesting that both alpha and beta subunits of Mac-1 heterodimer were released. Immunoprecipitation of control PMN lysates revealed bands of 198 kDa and 91 kDa slightly greater than those from the released Mac-1. An in vitro model of homotypic aggregation showed a similar loss of Mac-1 from PMNs; immunoprecipitates of the supernatant demonstrated peptide bands identical with those found in postischemic cardiac lymph. The appearance of soluble Mac-1 in vitro was prevented by anti-CD18 mAb, R15.7, and also by protease inhibition by PMSF. Thus, in vivo and in vitro, activated PMNs lose Mac-1 in a process that may be dependent upon adhesion and subsequent proteolysis.

To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.

We have previously shown that costimulation of endothelial cells with IL-1 + IL-4 markedly inhibits VCAM-1-dependent adhesion under flow conditions. We hypothesized that sialic acids on the costimulated cell surfaces may contribute to the inhibition. Northern blot analyses showed that Gal beta 1-4GlcNAc alpha 2, 6-sialyltransferase (ST6N) mRNA was up-regulated in cultured HUVEC by IL-1 or IL-4 alone, but that the expression was enhanced by costimulation, whereas the level of Gal beta 1-4GlcNAc/Gal beta 1-3GalNAc alpha2,3-sialyltransferase (ST3ON) mRNA was unchanged. Removing both alpha 2,6- and alpha 2,3-linked sialic acids from IL-1 + IL-4-costimulated HUVEC by sialidase significantly increased VCAM-1-dependent adhesion, whereas removing alpha 2,3-linked sialic acid alone had no effect; adenovirus-mediated overexpression of ST6N with costimulation almost abolished the adhesion, which was reversible by sialidase. The same treatments of IL-1-stimulated HUVEC had no effect. Lectin blotting showed that VCAM-1 is decorated with alpha 2,6- but not alpha 2,3-linked sialic acids. However, overexpression of alpha 2,6-sialyltransferase did not increase alpha 2,6-linked sialic acid on VCAM-1 but did increase alpha 2,6-linked sialic acids on other proteins that remain to be identified. These results suggest that alpha 2,6-linked sialic acids on a molecule(s) inducible by costimulation with IL-1 + IL-4 but not IL-1 alone down-regulates VCAM-1-dependent adhesion under flow conditions.

L-selectin enables capture and rolling of neutrophils on inflamed endothelium. This may facilitate the binding of agonists such as IL-8 and platelet-activating factor (PAF), which signal CD18-mediated firm adhesion and transmigration. Recent studies demonstrate that L-selectin can mediate transmembrane signaling. However, the functional effects of costimulation through agonist and L-selectin require further study. Here, we quantify cell adhesion, motility, and transmigration in response to co-activation through L-selectin and agonist. The surface expression of CD11b/CD18 increased and L-selectin decreased in proportion to the extent of L-selectin cross-linking. A flow cytometric assay was used to measure CD11b/CD18-dependent adhesion to fluorescent beads adsorbed with albumin. Neutrophil adhesion was detected within seconds of adding PAF (20 pM), IL-8 (50 pM), or cross-linking L-selectin. Costimulation through agonist and L-selectin potentiated by up to threefold the rate and extent of bead capture. Stimulation through L-selectin induced membrane ruffling, whereas PAF or IL-8 induced bipolar shape change. L-selectin cross-linking sustained the transient shape change induced by low concentrations (10-50 pM) of agonist. Chemokinesis stimulated by IL-8 was inhibited in the presence of cross-linking L-selectin. This was attributed to enhanced cell spreading following costimulation. Migration across HUVEC monolayers stimulated with IL-1 was also potentiated in the presence of L-selectin cross-linking. We propose that cross-linking of L-selectin and binding of agonist receptors may act synergistically to amplify neutrophil activation and emigration in the inflamed vasculature.

Since macromolecular permeability between endothelial cells is regulated by tight junctions (zonula occludens), we wished to determine whether they also regulate neutrophil transendothelial migration. HUVEC monolayers, a commonly used model for studying leukocyte transmigration, were characterized using electric cell substrate impedance sensing and transmission electron microscopy. We show that culture medium containing endothelial cell growth supplement (50 microg/ml) was sufficient and necessary for the development of endothelial tight junctions. The frequency with which tight junctions were observed by transmission electron microscopy was further increased (twofold) by culturing HUVEC monolayers in a 1:1 mixture of endothelial medium and astrocyte-conditioned medium. These astrocyte-conditioned HUVEC monolayers showed a >1.5-fold increase in transcellular electrical resistance. The extent of neutrophil migration across IL-1-treated (10 U/ml for 4 h) HUVEC monolayers was the same whether tight junctions were present or absent, and the molecular requirements for neutrophil transmigration (CD18 and intercellular adhesion molecule-1) were unaffected by culturing in astrocyte-conditioned medium. Immunostaining for proteins associated with the intercellular junctional domain (occludin, ZO-1, cadherin, beta-catenin, gamma-catenin, and platelet-endothelial cell adhesion molecule-1) was localized to the endothelial borders, regardless of the culture conditions. Discontinuities were observed in the border staining for occludin, ZO-1, cadherin, and beta-catenin at the tricellular corner where the borders of three endothelial cells intersected. Significantly, 75% of neutrophil migration across IL-1-treated HUVEC monolayers occurred at tricellular corners. It appears that neutrophils preferentially migrate around endothelial tight junctions by crossing at tricellular corners rather than passing through the tight junctions that lie between two endothelial cells.

Leumedins are small molecules that inhibit neutrophil movement into inflamed tissues. These compounds have been shown to inhibit the adherence of neutrophils in static adhesion assays mediated by beta2-integrins. We now report that leumedins, like activating agents, induce the loss of L-selectin from the neutrophil surface. The loss of L-selectin is unrelated to the inhibition of static adhesion, since neutrophils that have been pretreated with leumedins to cause shedding of L-selectin, followed by removal of drug, adhere normally in a static adhesion assay, and this adhesion is inhibited upon readdition of leumedin. In an assay of adhesion to endothelial cells under conditions of physiologic wall shear stress, leumedins prevent both primary capture of neutrophils mediated by L-selectin and firm adherence mediated by beta2-integrins.

Neutrophil emigration through endothelial cells under shear flow involves several adhesion processes including cell rolling, arrest, and transmigration. Rolling is mediated by selectins, while arrest and transmigration both require activated CD18 integrins. One mode of CD18 activation is via selectins expressed on neutrophils and endothelial cells. We have recently reported that cross-linking of L-selectin (CD62L) resulted in the rapid activation of CD18-dependent adhesion. In the current study, we examine whether binding of E-selectin (CD62E) and L-selectin can activate neutrophil CD18-dependent adhesion under shear flow. Human ICAM-1 (CD54) and E-selectin were co-transfected into L cells. Neutrophil capture, rolling, and arrest on these monolayers were quantitated in a parallel plate flow chamber at a wall shear stress of 2.0 dyne/cm2. Under these conditions, E-selectin supported cell capture and rolling on the monolayer, but did not trigger CD18-mediated cell arrest within 200 microm of rolling. However, when neutrophils were treated with anti-L-selectin mAb and cross-linked with a secondary mAb, approximately 50% of the cells arrested within 54 microm. Cell arrest was also observed in response to IL-8 stimulation. A subthreshold level of IL-8 in combination with L-selectin cross-linking potentiated the level of cell arrest due to either stimulus alone. The transition to cell arrest involved both LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18). Blocking either subunit alone failed to reduce arrest, while blocking both molecules with mAbs reduced the number to baseline levels. These data support the conclusion that L-selectin, but not E-selectin, can signal the transition from neutrophil rolling to cell arrest under shear flow.

To assess the potential contributions of endothelial vascular cell adhesion molecule-1 (VCAM-1) to leukocyte adhesion under conditions of flow, we evaluated the effects of cytokine stimulation of HUVEC on primary capture, rolling, and stable adhesion of mono- and lymphoblastoid cells in a parallel-plate flow chamber. Stimulation of HUVEC with IL-1beta or IL-4 for 24 h induced surface expression of VCAM-1, consistent with published results. Using blocking mAbs to inhibit specific adhesion molecules, we found that a significant level of primary capture at wall shear stresses between 0.5 and 2.0 dynes/cm2 could be attributed to alpha4 integrin and VCAM-1. This primary adhesion under flow was most often characterized by abrupt stationary adhesion with little evidence of rolling. Studies with IL-1beta-stimulated HUVEC revealed that domain 1 of VCAM-1 was solely responsible for alpha4 integrin-dependent primary capture under flow, but both domains 1 and 4 were utilized in alpha4 integrin-dependent adhesion under static conditions. In contrast to results obtained with these cytokines alone, stimulation of HUVEC with a combination of IL-1beta and IL-4 markedly reduced alpha4/VCAM-1-dependent adhesion under flow, even though the surface levels of VCAM-1 were greater than with either cytokine alone, and alpha4/VCAM-1-dependent adhesion under static conditions was intact. These results demonstrate that distinct functional differences exist between the interactions of VCAM-1 and alpha4 integrin under static and flow conditions, and they reveal the potential for endothelial modulation of adhesion under flow without altering VCAM-1-dependent adhesion under static conditions.

In this study, we examine neutrophil adhesion under flow conditions to cultured HUVEC stimulated for 4 or 24 h with IL-1. Interactions are measured using videomicroscopy and a parallel-plate flow system which is capable of distinguishing primary adhesion and rolling from secondary (firm) adhesion. We find that after 24 h, E-selectin does not contribute to primary adhesion, in contrast to a significant contribution after 4 h. Endothelial cell P-selectin does not contribute at either time point. Blocking or removing L-selectin from the neutrophil surface decreases adhesion 50 to 70% at either time point, and neuraminidase treatment of neutrophils decreases adhesion by 85%. These results suggest that after a 24-h stimulation, primary adhesion depends on a distinct selectin-like interaction in which a HUVEC receptor binds carbohydrates on neutrophil glycoproteins, including L-selectin. We also find that secondary adhesion in this system can be inhibited 90% with Ab blockade of CD18/ICAM-1 interactions after a 24-h stimulation, and that a combination of IL-1 and IL-4 selectively down-regulates the pathway for primary adhesion at 24 h. These results demonstrate that neutrophils adhere using different receptor pathways following 4- and 24-h stimulations, and provide experimental data characterizing some properties of the receptors involved. One of the pathways that is evident at 24 h appears to be a novel selectin-like interaction.

The mechanisms by which neutrophils migrate through the alveolar interstitium during acute lung inflammation are unknown. We wished to determine whether platelet-activating factor (PAF) and IL-8, two important mediators in neutrophil transendothelial migration, stimulated neutrophil adherence and motility on lung fibroblasts. Canine fibroblasts grown from lung explants were characterized by light and electron microscopy, and flow cytometry. Unstimulated neutrophils adhered poorly (less than 2%) to cultured fibroblasts. However, neutrophils stimulated with PAF (20-200 nM) showed a dose-dependent increase in adherence that was largely (70%) mediated by the beta 2 (CD11/CD18) integrins; adherence was less dependent (50%) on fibroblast intercellular adhesion molecule-1. Conversely, neutrophils stimulated with canine rIL-8 did not increase their adherence to fibroblasts. PAF-stimulated neutrophils were nonmotile on the surface of the fibroblast, but subsequent addition of rIL-8 (10(-8) M) induced motility that was entirely CD1 8 dependent. Fibroblasts stimulated with human rTNF-alpha or Escherichia coli endotoxin (LPS) were a significant source of IL-8 mRNA. In response to rTNF-alpha (50 U/ml), IL-8 mRNA was detected at 2 h by northern blot analysis; it peaked at 6 h and returned to baseline by 24 h. Fibroblasts stimulated with rTNF-alpha secreted IL-8 protein into the culture medium; secreted IL-8 was chemotactic for neutrophils. These data suggest that fibroblasts can function not only as an adhesive substrate, but also as a source of stimulation for neutrophil migration through the inflamed alveolar interstitium.

Emigration of leukocytes at sites of inflammation is initiated by the selectin family of carbohydrate-binding adhesion molecules. Molecular crossbridges initiate rolling of cells along the vascular endothelium where chemokines such as IL-8 and platelet activating factor (PAF) may be presented to their receptors on the leukocyte surface resulting in cell stimulation. Integrin activation appears to be a requirement for subsequent cell localization and diapedesis into the tissue. Several recent reports have demonstrated that ligation and cross-linking of neutrophil L-selectin results in neutrophil activation, including intracellular calcium release, superoxide production, and induction of mRNA for production of IL-8 and TNF-alpha. The purpose of this study was to examine whether ligation and cross-linking of L-selectin would specifically result in activation of beta 2-integrin-dependent adhesion. A fluorescence flow cytometric assay was developed that directly measures Mac-1-dependent cell adhesion. Fluorescent latex beads (2-microns diameter) were adsorbed with albumin or fibrinogen and added in excess to human neutrophils in a shear-stirred suspension. Following stimulation the kinetics of bead capture by neutrophils was continuously measured in real time on the flow cytometer. The onset of bead binding was detected in the presence of extremely low concentrations of PAF (10 pM) or formyl peptide (0.2 nM) stimulation. Ligation of L-selectin with whole IgG DREG200 or DREG56 Ab, but not controls (anti-CD44, -CD45, -CD11a), resulted in a significant potentiation of bead binding. Cross-linking F(ab')2 fragments of DREG200 with a goat anti-mouse F(ab')2 secondary Ab also stimulated beta 2-integrin-dependent adhesion in a dose-dependent fashion. A chimeric form of DREG200 expressing gamma 4 or gamma 1 isotypes of human Fc domain also stimulated cell adhesion when cross-linked. Surface expression of CD18 and an activation-dependent epitope, as detected with mAb24, also increased in response to L-selectin cross-linking. Cross-linking L-selectin induced significant adhesion and transmigration of neutrophils across human umbilical vein endothelial cells. We propose that cross-linking of L-selectin results in a cell signal that directly stimulates beta 2-integrin adhesive responses.

The effect of selective cell activation through L-selectin and Mac-1 molecules cross-linking on neutrophil superoxide anion (O2-) generation and changes in intracellular calcium [Ca2+]i was investigated. Cross-linking of L-selectin using different mAbs induced a rapid and transient increase in [Ca2+]i and O2- generation by neutrophils that were dependent on the extent of L-selectin cross-linking and mAb epitope binding. In addition, cross-linking of L-selectin induced an up-regulation of surface Mac-1 expression on neutrophils. Cross-linking of Mac-1 molecules with mAb also induced significant changes in [Ca2+]i levels and O2- generation by neutrophils, which was also dependent on the epitope binding by mAb. Pretreatment of neutrophils with LPS caused a marked decrease in the expression of L-selectin molecules and significant inhibition (i.e., 59 +/- 4%) of anti-L-selectin mAb-dependent O2- generation and [Ca2+]i signal. In contrast, LPS pretreatment caused a significant up-regulation of Mac-1 molecules on neutrophil and enhanced O2- generation by neutrophils. The data indicate that cross-linking of L-selectin and Mac-1 initiates changes in [Ca2+]i and O2- production in neutrophils and suggest that these distinct adhesion molecules independently may play important regulatory roles in modulating neutrophil-endothelial cell interactions, transmigration, and neutrophil function at sites of tissue injury.

Adenosine and adenosine analogues exhibit anti-inflammatory effects in vitro and in vivo, but their usefulness is limited by profound cardiovascular side effects. Therefore, we synthesized inhibitors of an enzyme involved in adenosine metabolism, adenosine kinase (AK) (EC 2.7.1.20), to enhance endogenous adenosine concentrations at sites of inflammation. GP-1-515 (4-amino-1-(5-amino-5-deoxy-1-beta-D- ribofuranosyl)-3-bromo-pyrazolo[3,4-d]pyrimidine), a novel AK inhibitor, decreased adhesion of activated human neutrophils to cultured endothelial cell monolayers by increasing local adenosine levels. The mechanism of inhibition in this assay seemed to involve selectin blockade and was independent of the beta 2 integrins. GP-1-515 and 2-chloroadenosine (a nonmetabolizable adenosine analogue) had no effect on the surface expression or shedding of adhesion molecules. An agent that disrupts the cytoskeleton, cytochalasin B, mimicked the effect of adenosine on cell adhesion. Interactions between L-selectin and the neutrophil cytoskeleton might be altered by adenosine and could contribute to adenosine-mediated adhesion inhibition.

CD18 is the common beta subunit for the heterodimeric leukocyte integrins that mediate many inflammatory cell adhesion responses including binding to intercellular adhesion molecules 1 and 2. CD18 deficiency in humans causes a severe granulocyte disorder with susceptibility to bacterial infections and high morbidity and mortality. Gene targeting was used to prepare an insertion mutation in the murine CD18 gene. The insertion mutation resulted in a hypomorphic rather than a null allele due to low expression from a cryptic promoter in the plasmid construct. Homozygous mutant mice are viable and fertile, demonstrate a mild granulocytosis, and have 2 or 16% of normal CD18 expression on granulocytes in the resting or activated state, respectively. Mutant mice show an impaired inflammatory response to a chemical peritonitis and delayed rejection of cardiac transplants. These CD18-mutant mice provide a model of the moderate form of the human disease and should be extremely valuable for in vivo analysis of the role of leukocyte integrin-dependent adhesion in inflammatory disease models.

The bolus i.v. infusion of cobra venom factor into rats results in acute lung injury that is neutrophil-dependent, oxygen radical-mediated, and requires CD18. In our studies a more precise definition regarding the role of beta-integrins and requirements for cytokines was obtained by the use of blocking antibodies. Lung injury was quantitated by changes in permeability (leakage of 125I-BSA) and hemorrhage (extravasation of 51Cr-RBC). In animals treated with anti-CD11a, the permeability and hemorrhage parameters were reduced by 30 and 29%, respectively. Treatment with anti-CD11b resulted in reductions in permeability and hemorrhage by 53 and 48%, respectively, whereas anti-intercellular adhesion molecule-1 reduced the parameters of injury by 60 and 75%, respectively. Not surprisingly, treatment with antibodies to very late Ag-4, TNF-alpha and IL-1 failed to show any protective effects, which contrasts to the requirements for these molecules in lung injury after deposition of IgG immune complexes. Protective interventions were associated with a reduction in lung content of myeloperoxidase. These studies indicate that, in the cobra venom factor model of acute lung injury in rats, engagement of Mac-1, lymphocyte function-associated Ag-1, and intercellular adhesion molecule-1 are essential, whereas, in contrast to other models of neutrophil mediated lung injury, cytokines (TNF-alpha and IL-1) and very late Ag-4 are not required for the full development of injury.

After intrapulmonary deposition of IgG or IgA immune complexes, injury has been recently shown to be CD18-dependent in both cases and E-selection-dependent only in the former case. In our studies further evaluation of the requirements for beta 1 and beta 2 integrins and intercellular adhesion molecule-1 (ICAM-1) has been undertaken. In the IgG immune complex model, which is neutrophil dependent, anti-CD11a reduced injury (as measured by changes in permeability and hemorrhage) by 61 and 43%, respectively, whereas a newly developed anti-CD11b produced minimal protection (16 and 19%, respectively). Treatment of rats with increasing doses (1.5- and 3.0-fold) of antibody to rat CD11b failed to demonstrate additional protective effects in this model of injury. Anti-ICAM-1 reduced the parameters of injury by 61 and 78%, respectively, while anti-VLA-4 reduced the injury parameters by 40 and 35%, respectively. There were reductions in lung content of myeloperoxidase, roughly corresponding to the protective effects of the interventions. In the IgA immune complex model of injury, in which lung macrophages appear to be the effector cells, anti-CD11a reduced the injury parameters (permeability and hemorrhage) by 36 and 33%, respectively, whereas anti-CD11b reduced the parameters of injury by 63 and 67%, respectively. In this model, anti-ICAM-1 reduced the parameters of injury by 61 and 56%, respectively, while anti-VLA-4 reduced the parameters by 77 and 62%, respectively. The cell content of bronchoalveolar lavage fluids revealed changes that have been shown to reflect protective interventions in both models of immune complex-induced injury. These findings suggest that, in IgG and IgA immune complex models of lung injury, both VLA-4 and ICAM-1 are required, although lymphocyte function-associated Ag-1 is the predominant beta 2 integrin requirement in the IgG immune complex-induced model of injury and Mac-1 is the predominant requirement for IgA immune complex-induced lung injury. Thus, engagement in the lung of adhesion molecules in a manner leading to injury depends on the nature of the inflammatory stimulus and the type of phagocytic cells involved in the development of injury.

Acute respiratory virus infections are often associated with an early influx of neutrophils (PMN) into the airways. Maximal cytoxic injury by PMN depends on tight cell-cell adhesion. Infection of some cell types by respiratory and other viruses has been shown to increase PMN adhesion to these cells by undefined mechanisms. We studied adhesion by human PMN to monolayers of primary (1 degree) human tracheal epithelial cells (TEC) or an immortalized cell line derived from human TEC, 9HTEo-, that had been infected with parainfluenza virus type 2 (PiV2). PMN adhesion to uninfected 1 degree TEC was very low (< 5%), but PMN adhesion to PiV2-infected 1 degree TEC was greatly increased (89 +/- 7%). PMN adhesion to 9HTEo- cells was 47 +/- 6%, but increased, 87 +/- 8%, for PiV2-infected 9HTEo- cells. Surface intercellular adhesion molecule-1 (ICAM-1) expression on 1 degree TEC, as determined by immunofluorescence flow cytometry, was relatively low (23 fluorescence units) but doubled by 24 h after PiV2 infection and tripled by 48 h. The 9HTEo- cells constitutively expressed higher levels of surface ICAM-1 (120 units) which did not increase with PiV2 infection. Treatment of non-PiV2-infected 9HTEo- cells with mAb (R6.5) to ICAM-1 reduced PMN adhesion to these cells from 47 +/- 8 to 23 +/- 5%. Identical mAb treatment of either 1 degree TEC or 9HTEo- cells infected with PiV2 had no significant effect on PMN adhesion. Treatment of the PMN with mAb against CD11a, CD11b, or CD18 markedly reduced PMN adhesion to PiV2-infected 1 degree TEC and 9HTEo- cells. We conclude that PiV2 infection of human TEC causes a marked increase in their adhesive interactions with PMN by inducing increased surface expression of both ICAM-1 and one or more, as yet uncharacterized, non-ICAM-1 adhesion molecules that function as counter-receptors for CD11/CD18 on PMN. These mechanisms of adhesion may play a role in epithelial damage during acute respiratory virus infections.

Acute lung injury produced by deposition of IgA immune complexes is complement-dependent, neutrophil-independent, oxygen radical-mediated, and may be a result of the formation of the hydroxyl radical (HO) generated directly or indirectly from activated lung macrophages. The current studies were designed to evaluate further the pathophysiologic events that occur after intrapulmonary deposition of IgA immune complexes. Pretreatment of rats with the human recombinant soluble complement receptor-1 resulted in marked attenuation of IgA immune complex-induced lung injury. Intravenous administration of antibody to CD18, but not antibody to CD11b, was highly protective against lung injury. Treatment of animals with either anti-endothelial leukocyte-adhesion molecule-1 or anti-TNF-alpha, both of which were highly protective against IgG immune complex-induced lung injury, had no protective effects in the model of IgA immune complex-induced lung injury. Immunohistochemical analysis revealed up-regulation of the endothelial leukocyte adhesion molecule-1 in the pulmonary vasculature after deposition of IgA immune complexes. This up-regulation was TNF-alpha-dependent. The arginine analog, NG-monomethyl-L-arginine, was highly protective against IgA immune complex-induced lung injury. This protective effect was reversed by the co-presence of L-arginine (but not D-arginine). Protective interventions against IgA immune complex-induced lung injury were inversely correlated with the numbers of macrophages that could be retrieved by lung lavage. These data suggest fundamental differences in the pathogenesis of lung injury after intrapulmonary deposition of IgA immune complexes, as compared with injury caused by deposition of IgG immune complexes. In the latter, neutrophils, intrapulmonary generation of TNF-alpha, and up-regulation of pulmonary vascular endothelial leukocyte-adhesion molecule-1 are required for the full development of lung injury, whereas no such requirements appear in the case of IgA immune complex-induced lung injury. Full expression of IgA immune complex-induced lung injury also appears to require L-arginine, suggesting a possible role for nitric oxide or its derivatives in events ultimately leading to injury.

The roles of beta 2 integrin molecules in neutrophil accumulation and tissue injury have been examined by the use of antibodies that are reactive with human CD11b and CD18 and cross-react with the homologous epitopes on rat neutrophils. Adherence to rat pulmonary artery endothelial cells by human neutrophils and endothelial cell killing by phorbol ester-activated human neutrophils required CD11b, CD11c, and CD18. Companion adherence studies between rat neutrophils and endothelial cells revealed a requirement for both CD11b and CD18. Neither anti-CD11b nor anti-CD18 depressed in vitro responses (O2- generation and chemotactic migration) of rat neutrophils. The accumulation of neutrophils in glycogen-induced peritoneal exudates was diminished substantially in rats treated with either anti-CD18 or anti-CD11b. In oxidant-mediated acute lung injury induced by rapid intravascular infusion of cobra venom factor, treatment of rats with either anti-CD18 or anti-CD11b significantly attenuated injury as assessed by increases in vascular permeability and hemorrhage. These protective effects correlated morphologically with diminished adhesion of neutrophils to interstitial intrapulmonary capillary endothelial cells. In studies of immune complex (BSA-anti-BSA)-induced alveolitis and dermal vasculitis, anti-CD18 had protective effects at all doses of anti-BSA employed. The protective effects of anti-CD18 correlated with diminished neutrophil accumulation in tissues at lower doses of anti-BSA. Although anti-CD11b was not effective under the same experimental conditions, intratracheal administration of this antibody conveyed protection against immune complex-induced lung injury, suggesting that both CD11b and CD18 are required for the full expression of injury. The current studies also demonstrated that when surface-bound IgG immune complexes were treated with fresh rat serum, the increment in O2- and TNF alpha generated by alveolar macrophages was suppressed by anti-CD18, but not by anti-CD11b, suggesting a heretofore unrecognized role for CD18 in the O2- and TNF-alpha responses of alveolar macrophages. Thus, neutrophil beta 2 integrins play a requisite role for the full expression of complement-dependent and oxygen radical-mediated injury of the lung and dermal vasculature.

The contributions of the canine neutrophil lectin adhesion molecule-1 (LECAM-1) (canine homologue of the murine MEL-14 Ag) in neutrophil-endothelial cell adhesion and transendothelial migration were studied using anti-LECAM-1 mAb, CL2/6, and SL1 under static conditions and at wall shear stresses of up to 1.85 dynes/cm2 (dpc). Both mAb were found to inhibit attachment of neutrophils to cytokine-stimulated canine jugular vein endothelium. The inhibitory effects of the anti-LECAM-1 mAb were more evident at a wall shear stress of 1.85 dpc (greater than 50%) than at 0.23 dpc or under static conditions (approximately 30%). In contrast the anti-CD18 mAb, R15.7, exhibited higher inhibitory ability at the lower shear stress and under static conditions with marginal inhibition of adhesion at 1.85 dpc. Anti-LECAM-1 and anti-CD18 mAb showed additive inhibitory effects at the lower wall shear stress and under static conditions. Chemotactic stimulation of the neutrophils caused rapid down-regulation of LECAM-1 from the neutrophil surface and reduced adhesion by 60% at a wall shear stress of 1.85 dpc. This inhibition was not additive to anti-LECAM-1 mAb. Pretreatment with CL2/6 or SL1 did not affect trans-endothelial migration of adherent neutrophils under any experimental conditions tested. Anti-CD18 mAb, however, blocked transendothelial migration by 98% and 56% under static condition and at a wall shear stress of 0.23 dpc, respectively. The results in this report indicate that canine LECAM-1 is involved in the initial adhesion of unstimulated neutrophils to cytokine-stimulated endothelial cells under flow, but in contrast to CD18-integrins, plays no role in the transendothelial migration.

To define further the molecular basis for abnormal interactions of cord blood or neonatal neutrophils with endothelial cells in vitro, we studied neutrophil adhesion and migration under experimental conditions specifically designed to evaluate CD18-independent mechanisms. Unstimulated cord blood neutrophils of healthy term neonates demonstrated significantly diminished adhesion to IL-1-stimulated endothelial cell monolayers under conditions of shear stress (congruent to 1.85 dynes/cm2); overall levels of migration by neonatal cells were also significantly diminished, although the adherent subpopulation of these cells migrated relatively normally. A mAb (DREG-56) against the human homologue of the murine MEL-14 antigen (termed lectin-, epidermal growth factor-, complement binding domain-cell adhesion molecule-1 (LECAM-1), a member of the LEC-CAM family of adhesion molecules) markedly inhibited adhesion of healthy adult but not cord blood neutrophils. In additional assessments of endothelial cell adhesion or migration in the absence of shear forces, cord blood neutrophils demonstrated significantly diminished values compared to adult controls. Moreover, mAb DREG-56 significantly diminished adhesion of healthy adult but not cord blood suspensions in the presence or absence of the anti-CD18 mAb R15.7. Immunofluorescence assessments of unstimulated cord blood neutrophils or neutrophils of neonates 12 to 48 h of age showed dramatically diminished levels of surface LECAM-1 compared to adult neutrophils. Chemotactic stimuli (FMLP, 10 nM, 15 min) consistently "down-regulated" surface LECAM-1 on adult neutrophils to levels approximately 10% of unstimulated suspensions and comparable to those of most unstimulated neonatal suspensions. Moreover, FMLP stimuli elicited little or no down-regulation of LECAM-1 on neonatal cells. In comparative studies, endothelial cell adhesion of unstimulated cord blood or adult control neutrophils (assessed under conditions of flow) was directly related to levels of neutrophil surface LECAM-1. Although FMLP stimulation significantly diminished both adhesion and LECAM-1 surface levels of adult control cells, the adhesion and LECAM-1 expression observed with cord blood cells were not significantly influenced by this stimulus. The mechanisms underlying diminished LECAM-1 expression and LECAM-1-dependent adhesion of neonatal neutrophils and the physiologic significance of these abnormalities deserve investigation.

Human neutrophils exposed to protein-coated polystyrene or cultured endothelial monolayers produce large quantities of H2O2 in response to soluble stimuli that elicit little or no secretion of reactive oxygen species from cells in suspension. To characterize the mechanisms involved in this adherence-dependent respiratory burst, we have investigated the possible role of one integrin known to participate in the adhesion of neutrophils to endothelial cells, CD11b/CD18 (Mac-1). H2O2 production was examined with chemotactic factor-stimulated human and canine neutrophils exposed to protein-coated surfaces and cultured human and canine endothelial cells. The two protein-coated surfaces used were type I collagen-coated glass or plastic, a surface to which neither human nor canine neutrophils adhered, and keyhole limpet hemocyanin (KLH)-coated glass or plastic, a surface to which human and canine neutrophils adhered only after chemotactic stimulation. FMLP-stimulated human neutrophils and platelet activating factor-stimulated canine neutrophils failed to produce detectable H2O2 when in contact with type I collagen, but secreted large amounts of H2O2 when adherent to KLH or endothelial cell monolayers. FMLP-stimulated neutrophils from patients with CD18-deficiency failed to adhere to any of these surfaces and failed to produce H2O2 under these conditions. mAb reactive with CD18 and CD11b were equally effective in markedly inhibiting the adhesion of normal human neutrophils to these surfaces and markedly inhibited the production of H2O2. A mAb reactive with CD18 blocked adhesion of stimulated canine neutrophils, and mAb directed against both CD18 and CD11b blocked H2O2 production by canine neutrophils on KLH and endothelium. A nonbinding mAb and a mAb reactive with CD11a did not inhibit H2O2 production of human cells on KLH or endothelial monolayers, and nonbinding and binding control mAb did not inhibit H2O2 production by canine neutrophils. These results indicate that Mac-1 (CD11b/CD18) can mediate adhesion-dependent H2O2 production by human and canine neutrophils exposed to chemotactic factors.

Human C1 inhibitor (C1 INH) enhances the chemotactic responsiveness of human leukocytes to lipopolysaccharide (LPS), AgAb complex, and zymosan-activated plasma or serum when added to the compartment of the chemotaxis chamber containing the cells. It seems to affect cells directly and causes an increased number of leukocytes to respond to the chemotactic factors at early time intervals. Spontaneous motility does not appear to be affected. Results of studies employing anti-C3 and anti-C5 sera seem to indicate that the chemotactic factor derived from C5 is involved in the C1 INH induced enhancement of chemotaxis. In fact, preliminary experiments utilizing trypsinactivated C3 as a chemotactic source indicate that C1 INH causes inhibition of chemotactic response to C3a. C1 INH is the first naturally occurring plasma component reported to cause enhanced chemotactic responsiveness.

The role of lymphocyte function-associated antigen 1 (LFA-1) in human T cell chemotaxis was investigated by using mAb specific to the beta-chain (TS1/18) (CD18) and alpha-chain (TS1/22) (CD11a) of LFA-1. T cell chemotaxis in response to IL-2 and to lymphocyte chemotactic factor (LCF) was markedly suppressed by the addition of TS1/18. TS1/22 was a less effective inhibitor than TS1/18 with only LCF stimulated responses showing significant inhibition when compared in seven different T cell preparations. Neither TS1/18 nor TS1/22 antibody inhibited random T cell migration. Control mAb to CD4 T cells failed to inhibit T cell random migration or chemotaxis. Additional studies to evaluate the adherence and migration of T cells through IL-1-stimulated human umbilical vein endothelial cell (HUVEC) monolayers showed that both TS1/22 and TS1/18 suppressed T cell migration through HUVEC, but failed to inhibit adherence of T cells to these cells. These studies indicate that LFA-1 plays a role in the migration of T cells through HUVEC and in the in vitro chemotactic response of T lymphocytes to IL-2 and LCF.

The 1A protein of respiratory syncytial (RS) virus is a small, 64-amino acid hydrophobic protein expressed in infected cells. We previously showed that the C-terminal domain of 1A contained a site for stimulation of RS virus-reactive Th lymphocytes in BALB/c and SJL/J mice. In this report we modeled a series of overlapping synthetic peptides of the 1A protein and we present evidence to suggest that the C-terminal domain of the 1A protein contains not one, but two, Th lymphocyte-stimulating sites. Although these sites are extensively overlapping they can be distinguished on the basis of presentation by distinct class II restriction elements of the murine MHC. Additionally, we present evidence to suggest that a 16-amino acid synthetic peptide which contains both of these T cell sites can stimulate T cell activity in mice of multiple different class II MHC haplotypes, perhaps representing a potential lead for designing a "universal" T cell stimulator.