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C W Smith

Publications and source records attributed to C W Smith.

At least 19 recordsLinked to original sources
PubMed1995

Induction of interleukin-6 synthesis in the myocardium. Potential role in postreperfusion inflammatory injury.

BACKGROUND: Neutrophil-induced injury of myocardial cells requires the expression of intercellular adhesion molecule-1 (ICAM-1) on the myocyte surface and is mediated by ICAM-1-CD11b/CD18 adhesion. We have previously shown that interleukin-6 (IL-6) cytokine activity, present in cardiac lymph, induces ICAM-1 on isolated cardiac myocytes. Furthermore, in previous in vivo studies, we have also shown ICAM-1 mRNA induction in the myocardium within the first hour of reperfusion in the previously ischemic viable zone. We hypothesized that induction of IL-6 synthesis in the myocardium was an integral part of the reaction to injury resulting from ischemia and reperfusion and was associated with induction of ICAM-1 on myocardial cells. METHODS AND RESULTS: In this study, cloned canine IL-6 cDNA was used as a molecular probe to study the regulation of IL-6 in an awake canine model of myocardial ischemia and reperfusion. IL-6 mRNA was induced in ischemic and reperfused segments of myocardium preferentially in segments previously exposed to severe ischemia. Peak levels of IL-6 mRNA were reached within 3 hours of reperfusion. At the same time, IL-6 mRNA and ICAM-1 mRNA were found in the same myocardial segments. In contrast to hearts that were ischemic for 1 hour and reperfused for 3 hours, nonreperfused hearts after 4 hours of persistent ischemia demonstrated minimal induction of ICAM-1 or IL-6 despite similar degrees of injury and blood flow reductions during ischemia. After 24 hours of persistent ischemia, levels of IL-6 mRNA were comparable to those observed in hearts that were ischemic for 1 hour and subsequently reperfused for 24 hours. CONCLUSIONS: Our results demonstrate induction of IL-6 mRNA in the myocardium and that this synthesis is accelerated by reperfusion. Evidence is also provided to show that peak IL-6 mRNA precedes that of ICAM-1 mRNA. These findings are compatible with our hypothesis that IL-6 is important in the induction of ICAM-1 in the area of ischemia. In addition, these studies suggest that the necessary factors to promote adhesive interactions between transmigrated neutrophils and cardiac myocytes are present in reperfused myocardium.

Amino Acid Sequence↗
PubMed1995

Peptides which bind to E-selectin and block neutrophil adhesion.

E-selectin is an inducible cell adhesion molecule which mediates rolling of neutrophils on the endothelium, an early event in the development of an inflammatory response. Inhibition of selectin-mediated rolling is a possible means for controlling inflammation-induced diseases, and several classes of compounds have been tested for this use. We describe here the use of recombinant peptide library screening for identification and optimization of novel ligands which bind to E-selectin. Several of these peptides bind with Kd values in the low nanomolar range and block E-selectin-mediated adhesion of neutrophils in static and flow-cell assays. Administration of the peptide to mice undergoing an acute inflammatory response reduced the extent of neutrophil transmigration to the site of inflammation, demonstrating the utility of this compound as a potential therapeutic. The identification of a peptide ligand for E-selectin suggests that the complete natural ligand for this adhesion molecule may include protein as well as carbohydrate moieties.

Amino Acid Sequence↗
PubMed1995

L-selectin (CD62L) cross-linking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) beta 2-integrin.

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.

Adult↗
PubMed1995

Interactions between the terminal bases of mammalian introns are retained in inosine-containing pre-mRNAs.

Nuclear pre-mRNA splicing has a fundamentally similar two-step mechanism to that employed by group II self-splicing introns. It is believed that nuclear pre-mRNA splicing involves a network of RNA-RNA interactions which form the catalytic core of the active spliceosome. We show here a non-Watson-Crick interaction between the first and last guanosine residues of a mammalian intron. As in Saccharomyces cerevisiae, substitution of the conserved guanosines at the 5' and 3' splice sites by A and C respectively, specifically suppresses step 2 splicing defects resulting from the individual mutations. No other combination of terminal nucleotides was able to restore splicing. We additionally provide independent evidence for an indirect interaction between other nucleotides of the consensus splice sites during step 2 of splicing. Substitution of the nucleotide in the +3 position of the 5' splice site affects competition between closely spaced AG dinucleotides at the 3' splice site, although the interaction is not via direct differential base pairing. Finally, we show that complete substitution of guanosine residues by inosine in a pre-mRNA has only a modest effect upon step 2 of splicing, although earlier spliceosome assembly steps are impaired. Predictions can thus be made about the precise configuration of the non-Watson-Crick interaction between the terminal residues.

Adenine↗
PubMed1995

The differential processing of homodimers of reverse transcriptases from human immunodeficiency viruses type 1 and 2 is a consequence of the distinct specificities of the viral proteases.

Active, recombinant p68 reverse transcriptase (RT) from human immunodeficiency virus type 2 (HIV-2), with an NH2-terminal extension containing a hexahistidine sequence was isolated from extracts of Escherichia coli by immobilized metal affinity chromatography. Treatment of the purified p68/p68 homodimer of HIV-2 RT with recombinant HIV-2 protease generates stable, active heterodimer (p68/p58) that is resistant to further hydrolysis. Analysis of this p68/p58 HIV-2 RT heterodimer revealed that while one subunit is intact p68, the p58 subunit is COOH-terminally truncated by cleavage, not at Phe440 as is seen in processing of the p66/p66 HIV-1 RT homodimer by HIV-1 protease, but at Met484. The expected COOH-terminal p10 fragment resulting from hydrolysis of p68 at Met484 is not released intact, but undergoes further cleavage at Asn494, Met503, and Tyr532. Processing of p68/p68 HIV-2 RT with the HIV-1 protease led to cleavage of the Phe440-Tyr441 bond, exactly as is seen with p66/p66 HIV-1 RT, to give the analogous p53 subunit. Studies of a peptide substrate modeled after residues 437-444 in HIV-2 RT showed that while the HIV-1 protease was able to cleave the Phe440 bond, this bond was resistant to cleavage by the HIV-2 enzyme. Our findings provide a rationale for the previous observation that the RT heterodimer isolated from HIV-2 lysates is larger than that from HIV-1. We conclude that the p68/p58 HIV-2 RT heterodimer, containing the Met484 truncated p58 subunit, is a biologically relevant form of the enzyme in vivo.

Amino Acid Sequence↗
PubMed1995

Activation of human neutrophils through L-selectin and Mac-1 molecules.

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.

Animals↗
PubMed1995

Inhibition of neutrophil adhesion by adenosine and an adenosine kinase inhibitor. The role of selectins.

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.

2-Chloroadenosine↗
PubMed1995

Characterization of human colon carcinoma variant cells selected for sialyl Lex carbohydrate antigen: liver colonization and adhesion to vascular endothelial cells.

The content of sialyl Lewis-X antigen (Neu5Ac alpha 2-3Gal beta 1-4(fuc alpha 1-3)GlcNAc-R: sialyl Le(x)) was previously shown to correlate with the progression of human colon carcinomas to the advanced stages. Variant cell lines with high (KM12-HX) or low (KM12-LX) levels of cell surface sialyl Le(x) were isolated from the heterogeneous KM12C cells to characterize the biological behavior of colon carcinoma cells with elevated cell surface contents of sialyl Le(x). When these cells were injected intrasplenically into nude mice, KM12-HX cells colonized to the liver more efficiently than KM12-LX cells. Under in vitro conditions, KM12-HX cells demonstrated greater degree of adhesion to cytokine-activated human umbilical vein endothelial cells than KM12-LX cells. The adhesion of KM12-HX cells was partially inhibited by antibodies specific for E-selectin, which was known to serve as a ligand for the sialyl Le(x) carbohydrate antigen. The treatment of KM12-HX cells with benzyl N-acetyl-alpha-D-galactosaminide, a putative inhibitor of the extension of O-linked carbohydrate chains, reduced the rate of adhesion. These results suggested that the interaction of endothelial cell surface E-selectin with O-linked carbohydrate chains on colon carcinoma cell surface glycoproteins played an important role in the adhesion.

Animals↗
PubMed1995

Group B streptococci elicit leukotriene B4 and interleukin-8 from human monocytes: neonates exhibit a diminished response.

Neonatal monocytes have diminished function compared with adult cells. The ability to recruit neutrophils through elaboration of chemoattractants has not been evaluated in humans. The pattern of chemoattractant release induced by group B streptococci (GBS) also is unknown. Adult and cord blood monocytes were stimulated with GBS. Supernatants were used as the attractant in blind well chambers; migration to neonatal supernatants was diminished. Interleukin-8 (IL-8) and leukotriene B4 (LTB4) were released in greater quantities by adult monocytes in response to either GBS or lipopolysaccharide. Opsonization of GBS was not required for IL-8 release. Adult monocytes released more LTB4 when stimulated with unopsonized GBS than with opsonized GBS; the neonate's LTB4 production did not increase. IL-8 and LTB4 accounted for the majority of chemoattractant activity released in response to GBS. Decreased production of LTB4 and IL-8 may contribute to the neonate's poor host response to GBS.

Adult↗
PubMed1995

Susceptibility to local and systemic bacterial infections in intercellular adhesion molecule 1-deficient transgenic mice.

The contribution of intercellular adhesion molecule 1 (ICAM-1) during systemic and local bacterial infections was studied in transgenic ICAM-1-deficient and control mice that were injected intraperitoneally (ip) or intradermally (id) with Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus. Mortality rates, blood cultures, white blood cell (WBC) counts and absolute neutrophil counts (ANCs) were obtained daily until cultures were sterile. Six and 24 h after injections, autopsies were done on randomly selected ip-inoculated mice and biopsies were done on randomly selected id-inoculated mice. Survival rates were similar. In ICAM-1-deficient mice, ip P. aeruginosa resulted in higher incidences of bacteremia at 24 h (P = .003) and 48 h (P = .002); id S. aureus resulted in larger skin lesions (P = .026). Leukocytosis persisted in ICAM-1-deficient mice 6 h after ip injection of E. coli; however, WBC counts and ANCs in peritoneal fluid did not differ. Although the inflammatory responses were similar histologically in ICAM-1-deficient and normal mice, differences in site- and stimulus-specific susceptibilities were noted.

Animals↗
PubMed1995

P-selectin- and CD18-mediated recruitment of canine neutrophils under conditions of shear stress.

Neutrophil mobilization at sites of inflammation or thrombosis involves the participation of several adhesion molecules expressed on neutrophils and vascular endothelial cells. Local vascular damage with disruption of the endothelium results in adhesion of platelets to the exposed subendothelium, and these platelets could also participate in neutrophil recruitment. This initial phase of mobilization could be followed by heterotypic aggregation to recruit more leukocytes in the area. The present study first examined the interactions of adherent canine platelets and flowing canine neutrophils using an in vitro system that simulates vascular flow conditions. Results showed that collagen-adherent platelets express the adhesion molecule P-selectin on their surface and can support neutrophil arrest (612 +/- 43 neutrophils/mm2) at shear stresses of approximately 2.5 dynes/cm2. Both transient adhesion (manifested by a rolling-type behavior) and complete arrest were observed. These interactions could be totally inhibited by a monoclonal antibody directed against platelet P-selectin (24 +/- 18 neutrophils/mm2) but not by a monoclonal antibody against neutrophil CD18 (625 +/- 46 neutrophils/mm2). Additionally, under shear mixing conditions (700 RPM), canine blood leukocytes exhibited aggregation (> 80% singlets recruited into aggregates after 5 minutes), and this process does not involve P-selectin but is dependent on the neutrophil integrin CD18. However, stimulation of the blood with platelet-activating factor (5-20 ng/ml) induced a rapid aggregation with a significantly greater number of aggregates when compared with stirring alone (68.3% +/- 3.2% versus 35.2% +/- 6.3% at 1 minute, P < 0.05), and this aggregation was both P-selectin and CD18 dependent. Overall, these two mechanisms of leukocyte recruitment (neutrophil arrest on adherent platelets and aggregation) could act sequentially and in a cooperative manner to bring into close contact platelets and neutrophils at sites of inflammation and thrombosis in pathologic conditions in the dog.

Animals↗
PubMed1995

A novel beta 1-dependent adhesion pathway on neutrophils: a mechanism invoked by dihydrocytochalasin B or endothelial transmigration.

It is generally accepted that the beta 2-integrin is restricted to mononuclear leukocytes. The objective of this study was to determine whether neutrophils can also express beta 1-integrin (specifically alpha 4 beta 1) and whether this can support neutrophil adhesion to endothelial cells and to extracellular matrix. We stimulated neutrophils with dihydrocytochalasin B (DHCB) and various chemotactic stimuli and observed that chemotactic stimuli induced neutrophil adhesion via beta 2-integrin (CD18), whereas DHCB and either fMLP, PAF, or IL-8 induced adhesion to endothelium or protein-coated plastic that was not inhibitable by anti-CD18 antibody. beta 2-integrin-deficient cells, which did not respond to chemotactic stimuli alone, also adhered avidly in the presence of chemotactic stimuli and DHCB. The induced neutrophil adhesion was inhibited by antibody to beta 1- or alpha 4-integrin chains, but only if an anti-beta 2-integrin antibody was also present. Flow cytometry revealed increased expression of both beta 1 and alpha 4 in the presence of fMLP plus DHCB. Transendothelial migration of neutrophils induced by chemotactic stimuli alone also increased expression of beta 1 and alpha 4. Transmigration across deendothelialized membranes induced a similar beta 1 expression on neutrophils suggesting that events other than an endothelial signal elicited beta 1-integrin expression. Transmigration-induced beta 1-dependent expression translated into only modest adhesion to protein-coated plastic. These data suggest that both a pharmacological (DHCB) and a physiological (transmigration) stimulus can invoke expression of alpha 4 and beta 1 on human neutrophils to mediate adhesion.

Antigens, CD↗
PubMed1995

Differential expression and release of CD54 induced by cytokines.

Intercellular adhesion molecule-1 (ICAM-1, CD54) is upregulated in many cell types stimulated by cytokines. A human hepatoblastoma cell line (C3A, a subclone of HepG2/C3 that is currently being used as a surrogate liver) and human lung adenocarcinoma cells (A549) were stimulated with interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), or IL-6 to determine any differences in cell type responsiveness to individual cytokines for ICAM-1 upregulation. Time courses were performed with each cytokine evaluating ICAM-1 mRNA, surface expression, and cICAM-1 in the cell culture media. Between 3 and 6 hours, IL-1 beta (30 U/mL) stimulated the greatest increase in hepatocyte ICAM-1 mRNA, followed by IFN gamma (100 U/ mL), and IL-6 (100 U/mL) in order of potency. Except for IL-6, cytokine-induced hepatocyte surface levels of ICAM-1 (immunofluorescence flow cytometry, mAb R6.5) were dose dependent, with inhibition at higher concentration. Highest levels followed stimulation with INF gamma (P < .05). Significantly less was found after both IL-1 beta and TNF alpha; none was detected after IL-6 (P < .05). In contrast, IL-1 beta stimulated significantly more cICAM-1 release from hepatocytes than the other cytokines (P < .001), and IL-6 stimulated modest cICAM-1. Between 3 and 6 hours in the A549 cells, IL-1 beta stimulated the greatest increase in ICAM-1 mRNA, followed by TNF alpha. Both responses were greater than that observed in the hepatocytes. IFN gamma- and IL-6-induced ICAM-1 mRNA synthesis was not different from unstimulated A549 cells. Cytokine-induced A549 surface levels of ICAM-1 (immunofluorescence flow cytometry, mAb R6.5) was highest for IL-1 beta (peak levels similar to hepatocyte response), modest with TNF alpha (peak levels less than hepatocytes), detectable with IFN gamma (much less than hepatocytes), and nondetectable after IL-6. No ICAM-1 release from A549 cells was induced under any condition. In hepatocytes the amount of ICAM-1 mRNA was best accounted for by considering both cell surface levels of ICAM-1 and cICAM-1 levels. In human lung adenocarcinoma cells, the cytokine induction of ICAM-1 mRNA could potentially be accounted for by observing cell surface levels of ICAM-1 because no cICAM-1 was produced. These results suggest that surface ICAM-1 and cICAM-1 may be differentially controlled by each cytokine and by each parenchymal cell type.

Adenocarcinoma↗
PubMed1994

CD18-dependent and L-selectin-dependent neutrophil emigration is diminished in neonatal rabbits.

Human neonatal neutrophils manifest decreases in mobility, adherence, and emigration compared with adult neutrophils that may contribute to the increased susceptibility of neonates to infection. In a developmental rabbit model, we show a reduced ability of neutrophils from 1-day-old rabbit pups to emigrate to inflamed peritoneium (3.7 +/- 0.35 x 10(6) neutrophils/mL peritoneal exudate) compared with 14-day-old (8.5 +/- 0.7 x 10(6)/mL) and adult rabbits (9.4 +/- 1.4 x 10(6) mL, P < .05) despite significantly increased blood neutrophil counts. Because the reductions in functional Mac-1 (CD11b/CD18) as well as the amount of surface L-selectin are hypothesized to be primarily responsible for the differences in human neonatal neutrophil mobility, we examined CD11b/CD18 and L-selectin in our model. Using flow cytometric analysis we found that similar to human neonates, neutrophils from 1-day-old rabbit pups had 57% of adult rabbit levels of L-selectin and, in contrast with adults, failed to show significant decreases in L-selectin after chemotactic stimulation. In addition, neutrophils from 1-day-old pups compared with adults showed a significantly diminished capacity to upregulate CD11b/CD18 after chemotactic stimulation in vitro, or after emigration to the inflamed peritoneum. Systemic administration of anti-L-selectin monoclonal antibody (MoAb) resulted in significant reduction in peritoneal neutrophils in adult (47%, P < .05) and 14-day-old rabbits (47%, P < .05), but was without effect in 1-day-old rabbits. Administration of anti-CD18 MoAb resulted in significant reduction in peritoneal neutrophil accumulation in all age groups though less in 1 day and 14 day (58% and 65%, respectively) than in adults (91%, P < .05). Only in the 14-day-old rabbits was there an additive effect of anti-L-selectin and anti-CD18 MoAbs compared with anti-CD18 alone (84% v 65%, P < .05). The findings in this in vivo rabbit model support the hypothesis that the previously described in vitro defects in human neonatal L-selectin and CD11b/CD18 may be major contributors to human neonatal inflammatory deficits.

Age Factors↗
PubMed1994

A beam profile generation algorithm for wedged half-beam blocked asymmetric fields.

A method is presented to predict beam profiles and outputs for wedged asymmetric fields defined by independent collimator jaws allowing fast generation of beam profiles whilst requiring very little additional data to that already used by most treatment planning systems in generating symmetric field isodoses. Symmetric field data are modified by the use of wedged primary off-centre ratios (POCRS) which are obtained from in air measurements of the largest possible wedged field. Beam hardening occurring within the flattening filter and wedge is taken into account by the use of attenuation coefficients measured under each wedge and used to generate the wedged POCR at depth. A full investigation into the comparison between measured and calculated profiles was performed which demonstrates favourable agreement across the whole of the asymmetric field including the penumbra.

Algorithms↗
PubMed1994

P-selectin and ICAM-1-dependent adherence reactions: role in the genesis of postischemic no-reflow.

The aim of this study was to determine whether immunoneutralization of P-selectin or intercellular adhesion molecule-1 (ICAM-1) (endothelial cell adhesion molecules involved in leukocyte rolling and firm adhesion, respectively) would attenuate the development of postischemic capillary no-reflow. Microvascular patency was assessed in vascularly isolated canine gracilis muscles by perfusion with contrast media (India ink) at the end of the experimental protocol. Computerized video imaging was used to quantitate the number of ink-containing microvessels (< 10 microns diam) per muscle fiber in histological samples obtained from isolated canine gracilis muscles subjected to 4.5 h of continuous perfusion (nonischemic control), 4 h of ischemia and 30 min of reperfusion (I-R), I-R + P-selectin monoclonal antibodies (MAbs) (MD6 or PB1.3), and I-R + ICAM-1 MAbs (CL18/6C7 or R6.5). The efficacy of a P-selectin MAb (MD3) that binds to a nonfunctional epitope was also evaluated. I-R was associated with a marked reduction in the number of patent capillaries per fiber (3.1 +/- 0.2 vs. 1.1 +/- 0.2 patent capillaries/fiber for control and I-R, respectively). Immunoneutralization with MAbs directed against functional epitopes on P-selectin (MD6 or PB1.3) significantly improved capillary perfusion (2.3 +/- 0.3 and 3.6 +/- 0.6 patent capillaries/fiber, respectively). On the other hand, MAb MD3, which binds to nonfunctional epitopes on P-selectin, failed to limit the development of postischemic no-reflow (1.0 +/- 0.2 patent capillaries/fiber). Immunoneutralization of ICAM-1 with CL18/6C7 and R6.5 increased the number of patent capillaries per fiber to 1.8 +/- 0.1 and 2.5 +/- 0.3, respectively. These data indicate that P-selectin and ICAM-1-dependent adherence reactions play an important role in the development of the no-reflow phenomenon in postischemic skeletal muscle.

Animals↗
PubMed1994

Induction of intercellular adhesion molecule-1 by lipopolysaccharide in canine alveolar macrophages.

Previous studies have demonstrated that alveolar macrophages (AMphis) adherent to plastic release interleukin-8 in response to lipopolysaccharide (LPS). We sought to determine whether LPS could also alter surface adhesion molecule expression and thus modulate additional AMphi adhesive interactions. Canine AMphis obtained by bronchoalveolar lavage of excised lung were adhered onto tissue culture plastic and exposed to LPS (0.01 to 100 ng/ml). Expression of beta 2 integrins and intercellular adhesion molecule-1 (ICAM-1) was subsequently determined by flow cytometry, a cDNA probe to canine ICAM-1 was used to quantify ICAM-1 mRNA, and changes in adhesion molecule function were assessed by evaluating the extent of homotypic aggregation. ICAM-1 and CD11a/CD18 were present on freshly isolated AMphis. CD11b/CD18 and CD11c/CD18 were expressed at lower levels. Nonadherent AMphis expressed a pattern of beta 2 integrin and ICAM-1 comparable to adherent cells. During short-term LPS stimulation (3 h), adherent AMphis increased both the synthesis and expression of ICAM-1. CD18 expression was either decreased or remained unchanged with LPS stimulation. LPS stimulation in vitro (> 0.01 ng/ml) enhanced the homotypic aggregation of adherent AMphis. Aggregation was blocked by monoclonal antibodies to ICAM-1 (CL18/6) and CD11a (R7.1) and CD18 (R15.7). Similar kinetics were found for expression of ICAM-1 and homotypic aggregation, suggesting that up-regulation of ICAM-1 is a major determinant of the LPS-stimulated aggregation of AMphis.

Animals↗