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Biomedical subjects

C W Smith

Publications and source records attributed to C W Smith.

At least 73 records · Page 4Linked to original sources
PubMed1999

Chimeric 7E3 Fab (ReoPro) decreases detectable CD11b on neutrophils from patients undergoing coronary angioplasty.

OBJECTIVES: The purpose of this study was to monitor the effects of chimeric 7E3 Fab (ReoPro) on leukocyte and platelet activation and interaction during coronary angioplasty. BACKGROUND: Increased expression of CD11b on monocytes and neutrophils promotes their adhesion to endothelial cells, extracellular matrix and smooth muscle cells. Thrombin-activated platelets adhere via P-selectin to monocytes and neutrophils. These cell interactions may affect the outcome of coronary angioplasty. METHODS: During coronary angioplasty, venous blood was obtained for flow cytometric detection of leukocyte CD11b; platelet CD41a, CD61a and CD62P; the percentage of leukocytes with adherent platelets and the intensity of bound platelet fluorescence. RESULTS: Leukocyte CD11b expression increased after angioplasty in control patients (neutrophils 171+/-25 to 255+/-31 mean fluorescence intensity [MFI, mean+/-SEM], n=25, p < 0.0001; monocytes 200+/-40 to 248+/-36 MFI, n=17, p < 0.05) and decreased in the patients selected to receive chimeric 7E3 Fab (neutrophils 146+/-30 to 82+/-22 MFI, n=25, p < 0.0001; monocytes 256+/- 53 to 160+/-38 MFI, n= 17, p < 0.05). Neutrophil CD11b decreased after in vitro incubation of whole blood with chimeric 7E3 Fab (n=5, p=0.01), but fMLP-induced increases in CD11b were not prevented. The CD11b expression was unchanged and increased with fMLP stimulation after in vitro incubation of isolated neutrophils with chimeric 7E3 Fab. Direct-labeled chimeric 7E3 Fab was not detected bound to neutrophils in whole blood or isolated cells using flow cytometric techniques. Adhesion of isolated neutrophils to protein-coated glass was not prevented by in vitro incubation with chimeric 7E3 Fab. Platelet activation increased after angioplasty in control patients (CD62P 8.9+/-0.8 to 12.3+/-1.2 MFI, n=25, p < 0.05; CD41a 382+/-25 to 454+/-26 MFI, n=25, p < 0.05, CD61a 436+/-52 to 529+/-58 MFI, n=11, p < 0.05); it did not increase in the patients selected to receive chimeric 7E3 Fab (CD62P 13.2+/-1.0 to 9.0+/-0.9 MFI, n=25, p < 0.05; CD61a 398+/-32 to 410+/-38 MFI, n=7, p=NS). Leukocytes with adherent platelets tended to increase in the control group of patients and decrease after the procedure in patients selected to receive chimeric 7E3 Fab; individual and procedure-related variability were marked. CONCLUSIONS: Despite standard aspirin and heparin therapy, leukocyte and platelet activation with platelet adherence to leukocytes occurs after coronary angioplasty. Although chimeric 7E3 Fab does not bind to leukocytes directly, it influences CD11b expression in whole blood. Modulation of platelet and leukocyte activation and interaction by chimeric 7E3 Fab may contribute to an improved outcome after coronary angioplasty.

Abciximab↗
PubMed1999

Effects of IGF-I, IGF-II, bFGF and PDGF on the initiation of mRNA translation in C2C12 myoblasts and differentiating myoblasts.

In order to study the mechanisms by which growth factors stimulate protein synthesis, C2C12 myogenic cells were treated with a variety of growth factors and the recruitment of free ribosomes to polysomes was quantified. All experiments were conducted on C2C12 myoblasts (24 h prior to induction of fusion) and differentiating myoblasts (24 h after induction of fusion). After the 2 h incubation, cells were rinsed with phosphate buffered saline and quickly frozen at -80 degrees C. Cell lysates were fractionated on 15-60% sucrose gradients by centrifugation at 200,000 x g for 1 h. Absorbance at 254 nm was recorded continuously across the gradient. The response to each of the four growth factors, IGF-I and-II, basic fibroblast growth factor (FGF), and platelet-derived growth factor was a decrease (P < 0.05) in monosome peak height and a increase (P < 0.05) in polysome percentage (P < 0.05). All responses were linear, except IGF-I, and the monosome peak height response to FGF which were quadratic (P < 0.05). None of the growth factors had a significant effect (P > 0.05) on RNA concentrations over the 2-h incubation. Protein content did not vary due to growth factor or level of treatment. This corroborates the hypothesis that the acute increase of protein synthesis exhibited by growth factor treated cells is due to an increase in the activity of existing ribosomes rather than an increase in ribosome synthesis. These results suggest that we can study the mechanisms regulating protein synthesis in muscle cells effectively by studying shifts in ribosomal activity. This method gave more consistent results than the H3-tyrosine incorporation and has the added benefit of not requiring the use of radioactivity. The strong correlation between monosome peak heights and percentage polysomes will allow researchers to measure total protein synthetic activity in a culture from the free or cytoplasmic fraction and to reserve the polysomes for other uses. The similarity of response among the various growth factors may indicate a common mechanism for increasing the initiation of protein synthesis.

Animals↗
PubMed1999

Effects of serum deprivation, insulin and dexamethasone on polysome percentages in C2C12 myoblasts and differentiating myoblasts.

An increase in the rate of protein synthesis in living cells can be achieved by regulating the quantity of mRNA, ribosomes, and enzymes available for translation or by regulating the efficiency at which existing components are used. Efficiency can be measured by comparing the number of ribosomes actively engaged in the synthesis of protein (polysomes) to the pool of free ribosomes. The objective of this study was to determine the percentage of ribosomes found as polysomes in C2C12 cells deprived of serum or exposed to insulin or dexamethasone 24 h before and after being stimulated to differentiate. Individual 60 mm culture dishes were exposed to serum-free control medium, medium containing serum, insulin, or dexamethasone for a period of 1 h or 2 h and then quickly frozen. The ribosomes and polysomes from these cells were separated by ultracentrifugation on 15 to 60% sucrose gradients and the absorbance across the gradient at 254 nm was recorded. Polysome percentages were determined as the area under the polysome peak divided by the total area under the curve. Serum deprivation caused a 12% decline in the percentage of ribosomes found as polysomes (P < 0.01). Dexamethasone caused a quadratic decline (P < 0.05) in polysome percentage, while insulin yielded a quadratic increase (P < 0.05). Protein synthesis assays measuring 3H-tyrosine uptake showed similar responses. These changes occurred in the absence of any differences in total RNA concentration. It was concluded that differentiation and the absence of serum in the media reduced the rate of recruitment of ribosomes for protein synthesis. Insulin increased ribosome recruitment which was also observed by a similar increase in incorporation of radio-labeled tyrosine.

Animals↗
PubMed1999

Transesophageal echocardiographic visualization of left ventricular malpositioned pacemaker electrodes: implications for lead extraction procedures.

Two cases of malpositioned LV electrodes are presented. Transesophageal echocardiography (TEE) allowed for a careful inspection of left-sided leads and for tracking their course. One LV and two right-sided leads were safely retrieved with TEE monitoring. One chronic LV lead was left in place as it was heavily fibrosed. TEE was helpful in the inspection and monitoring of the extraction and also in guided traction efforts. This is the first published report of echocardiographic visualization of the lead retrieval procedure.

Adult↗
PubMed1999

Polypyrimidine tract binding protein functions as a repressor to regulate alternative splicing of alpha-actinin mutally exclusive exons.

The smooth muscle (SM) and nonmuscle (NM) isoforms of alpha-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat alpha-actinin genomic clone encompassing the mutually exclusive exons was isolated and sequenced. The SM exon was found to utilize two branch points located 382 and 386 nucleotides (nt) upstream of the 3' splice site, while the NM exon used a single branch point 191 nt upstream. Mutually exclusive splicing arises from the proximity of the SM branch points to the NM 5' splice site, and this steric repression could be relieved in part by the insertion of spacer elements. In addition, the SM exon is repressed in non-SM cells and extracts. In vitro splicing of spacer-containing transcripts could be activated by (i) truncation of the transcript between the SM polypyrimidine tract and exon, (ii) addition of competitor RNAs containing the 3' end of the actinin intron or regulatory sequences from alpha-tropomyosin (TM), and (iii) depletion of the splicing extract by using biotinylated alpha-TM RNAs. A number of lines of evidence point to polypyrimidine tract binding protein (PTB) as the trans-acting factor responsible for repression. PTB was the only nuclear protein observed to cross-link to the actinin RNA, and the ability of various competitor RNAs to activate splicing correlated with their ability to bind PTB. Furthermore, repression of alpha-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA could be specifically restored by the addition of recombinant PTB. Thus, alpha-actinin mutually exclusive splicing is enforced by the unusual location of the SM branch point, while constitutive repression of the SM exon is conferred by regulatory elements between the branch point and 3' splice site and by PTB.

Actinin↗
PubMed1999

Pulmonary oligemia in aortic valve disease.

PURPOSE: To determine whether the severity of the radiographic appearance of oligemia correlates with the severity of cardiac dysfunction. MATERIALS AND METHODS: Nine readers graded a set of 25 chest radiographs (15 cases of aortic valve disease [AVD], 10 control cases without AVD) for blood volume and ventricular size. Blood volume was graded on a scale of -3 (severe hyperemia) to 0 (normovolemia) to +3 (severe oligemia). Ventricular size was graded on a scale of 0 (normal) to 3 (massively enlarged). The oligemia and ventricular size grades were added to yield the radiographic severity index. Pulmonary capillary wedge pressure, pulmonary arterial pressure, stroke volume, and cardiac output were measured at the time of catheterization. RESULTS: The five more experienced readers achieved good nonchance agreement (kappa = 0.48; P < .001). They were unanimous in scoring 12 cases as oligemic; variations occurred only in severity assessments. Oligemia was due to emphysema in one case and to AVD in 11. In oligemic cases, radiographic severity correlated significantly with wedge pressure (r = 0.93, P < .001) and pulmonary arterial pressure (r = 0.93, P < .002). CONCLUSION: Many cases of AVD show oligemia. The severity of oligemia correlates well with hemodynamic abnormality. Oligemia may be caused by atrial-pulmonary-vascular reflex vasoconstriction, low right ventricular output, and possibly high levels of atrial natriuretic factor.

Adult↗
PubMed1999

Expression of proinflammatory and proangiogenic cytokines in patients with head and neck cancer.

Altered immune, inflammatory, and angiogenesis responses are observed in patients with head and neck squamous cell carcinoma (HNSCC), and many of these responses have been linked with aggressive malignant behavior and a decrease in prognosis. In this study, we examined the hypothesis that HNSCC cells produce cytokines that regulate immune, inflammatory, and angiogenesis responses. We identified important regulatory cytokines in supernatants of well-defined and freshly cultured HNSCC cell lines by ELISA and determined whether these cytokines are detected in tumor cell lines and tissue specimens by immunohistochemistry. The serum concentration of the cytokines and cytokine-dependent acute phase inflammatory responses (i.e., fibrinogen, C-reactive protein, and erythrocyte sedimentation rate) from patients with HNSCC was determined, and the potential relationship of serum cytokine levels to tumor volume was analyzed. Cytokines interleukin (IL)-1alpha, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor were detected in similar concentration ranges in the supernatants of a panel of established University of Michigan squamous cell carcinoma (UM-SCC) cell lines and supernatants of freshly isolated primary HNSCC cultures. Evidence for the expression of IL-1alpha, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, and VEGF in HNSCC cells within tumor specimens in situ was obtained by immunohistochemistry. In a prospective comparison of the cytokine level and cytokine-inducible acute-phase proteins in serum, we report that cytokines IL-6, IL-8, and VEGF were detected at higher concentrations in the serum of patients with HNSCC compared with patients with laryngeal papilloma or age-matched control subjects (at P < 0.05). The serum concentrations of IL-8 and VEGF were found to be weakly correlated with large primary tumor volume (R2 = 0.2 and 0.4, respectively). Elevated IL-1- and IL-6-inducible acute-phase responses were also detected in cancer patients but not in patients with papilloma or control subjects (at P < 0.05). We therefore conclude that cytokines important in proinflammatory and proangiogenic responses are detectable in cell lines, tissue specimens, and serum from patients with HNSCC. These cytokines may increase the pathogenicity of HNSCC and prove useful as biomarkers or targets for therapy.

Acute-Phase Reaction↗
PubMed1998

Co-transcriptional commitment to alternative splice site selection.

Production of mRNA in eukaryotic cells involves not only transcription but also various processing reactions such as splicing. Recent experiments have indicated that there are direct physical connections between components of the transcription and processing machinery, supporting previous suggestions that pre-mRNA splicing occurs co-transcriptionally. Here we have used a novel functional approach to demonstrate co-transcriptional regulation of alternative splicing. Exon 3 of the alpha-tropomyosin gene is specifically repressed in smooth muscle cells. By delaying synthesis of an essential downstream inhibitory element, we show that the decision to splice or repress exon 3 occurs during a limited window of opportunity following transcription, indicating that splice site selection proceeds rapidly after transcription.

Alternative Splicing↗
PubMed1998

Fragment reconstruction and bone plate fixation versus bridging plate fixation for treating highly comminuted femoral fractures in dogs: 35 cases (1987-1997).

OBJECTIVE: To compare fragment reconstruction and bone plate fixation versus bridging plate fixation for treating highly comminuted (> 4 fragments) femoral fractures in dogs. DESIGN: Retrospective study. ANIMALS: 35 dogs with highly comminuted fractures of the femur. PROCEDURE: Medical records of all dogs included in this study were reviewed. Dogs had been treated with fragment reconstruction and bone plate application (n = 20) or major fragment alignment and bridging plate fixation (15). Postoperative and follow-up radiographs were evaluated. Operating and hospitalization times, bone alignment, bone healing, and complications were considered. RESULTS: There were no differences in hospitalization times, limb alignment, and complications between dogs with fractures treated with fragment reconstruction and dogs with fractures treated with bridging plate fixation. Dogs with fractures treated with bridging plate fixation had shorter operative times and faster times to radiographic evidence of bone healing. CLINICAL IMPLICATIONS: Bridging plate fixation is quicker to perform and results in faster healing than fragment reconstruction and bone plate fixation when used to treat comminuted femoral fractures.

Age Factors↗
PubMed1998

Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin.

BACKGROUND: After activation, platelets adhere to neutrophils via P-selectin and beta2-integrin. The molecular mechanisms and adhesion events in whole blood exposed to venous levels of hydrodynamic shear in the absence of exogenous activation remain unknown. METHODS AND RESULTS: Whole blood was sheared at approximately 100 s(-1). The kinetics of neutrophil-platelet adhesion and neutrophil aggregation were measured in real time by flow cytometry. P-selectin was upregulated to the platelet surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation increased linearly with platelet adhesion to neutrophils. Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhibition of neutrophil-platelet adhesion (approximately 30%) and neutrophil aggregation (approximately 70%). The residual amount of neutrophil adhesion was blocked with anti-CD11b/CD18. Treatment of blood with prostacyclin analogue ZK36374, which raises cAMP levels in platelets, blocked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK36374 and anti-CD18. Electron microscopic observations of fixed blood specimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated activation. CONCLUSIONS: The results are consistent with a model in which venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to mediate neutrophil aggregation. Abrogation of platelet adhesion and aggregation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. The described mechanisms are likely of key importance in the pathogenesis and progression of thrombotic disorders that are exacerbated by leukocyte-platelet aggregation.

Abciximab↗
PubMed1998

Hydrodynamic shear shows distinct roles for LFA-1 and Mac-1 in neutrophil adhesion to intercellular adhesion molecule-1.

The binding of neutrophil beta2 integrin to intercellular adhesion molecule-1 (ICAM-1) expressed on the inflamed endothelium is critical for neutrophil arrest at sites of tissue inflammation. To quantify the strength and kinetics of this interaction, we measured the adhesion between chemotactically stimulated neutrophils and ICAM-1-transfected mouse cells (E3-ICAM) in suspension in a cone-plate viscometer at shear rates typical of venular blood flow (100 s-1 to 500 s-1). The kinetics of aggregation were fit with a mathematical model based on two-body collision theory. This enabled estimation of adhesion efficiency, defined as the probability with which collisions between cells resulted in firm adhesion. The efficiency of beta2-integrin-dependent adhesion was highest ( approximately 0.2) at 100 s-1 and it decreased to approximately zero at 400 s-1. Both LFA-1 and Mac-1 contributed equally to adhesion efficiency over the initial 30 seconds of stimulation, but adhesion was entirely Mac-1-dependent by 120 seconds. Two hydrodynamic parameters were observed to influence integrin-dependent adhesion efficiency: the level of shear stress and the intercellular contact duration. Below a critical shear stress (<2 dyn/cm2), contact duration predominantly limited adhesion efficiency. The estimated minimum contact duration for beta2-integrin binding was approximately 6.5 ms. Above the critical shear stress (>2 dyn/cm2), the efficiency of neutrophil adhesion to E3-ICAM was limited by both the contact duration and the tensile stress. We conclude that at low shear, neutrophil adhesion is modulated independently through either LFA-1 or Mac-1, which initially contribute with equal efficiency, but differ over the duration of chemotactic stimulation.

Animals↗
PubMed1998

Stem cell factor induction is associated with mast cell accumulation after canine myocardial ischemia and reperfusion.

BACKGROUND: Myocardial infarction is associated with an intense inflammatory reaction leading to healing and scar formation. Because mast cells are a significant source of fibrogenic factors, we investigated mast cell accumulation and regulation of stem cell factor (SCF), a potent growth and tactic factor for mast cells, in the healing myocardium. METHODS AND RESULTS: Using a canine model of myocardial ischemia and reperfusion, we demonstrated a striking increase of mast cell numbers during the healing phase of a myocardial infarction. Mast cell numbers started increasing after 72 hours of reperfusion, showing maximum accumulation in areas of collagen deposition (12.0+/-2.6-fold increase; P<0.01) and proliferating cell nuclear antigen (PCNA) expression. The majority of proliferating cells were identified as alpha-smooth muscle actin-positive myofibroblasts or factor VIII-positive endothelial cells. Mast cells did not appear to proliferate. Using a nuclease protection assay, we demonstrated induction of SCF mRNA within 72 hours of reperfusion. Immunohistochemical studies demonstrated that a subset of macrophages was the source of SCF immunoreactivity in the infarcted myocardium. SCF protein was not found in endothelial cells and myofibroblasts. Intravascular tryptase-positive, FITC-avidin-positive, CD11b-negative mast cell precursors were noted in the area of healing and in the cardiac lymph after 48 to 72 hours of reperfusion. CONCLUSIONS: Mast cells increase in number in areas of collagen deposition and PCNA expression after myocardial ischemia. The data provide evidence of mast cell precursor infiltration into the areas of cellular injury. SCF is induced in a subset of macrophages infiltrating the healing myocardium. We suggest an important role for SCF in promoting chemotaxis and growth of mast cell precursors in the healing heart.

Animals↗
PubMed1998

Resident cardiac mast cells degranulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion.

BACKGROUND: Neutrophil-induced cardiomyocyte injury requires the expression of myocyte intercellular adhesion molecule (ICAM)-1 and ICAM-1-CD11b/CD18 adhesion. We have previously demonstrated interleukin (IL)-6 activity in postischemic cardiac lymph; IL-6 is the primary stimulus for myocyte ICAM- 1 induction. Furthermore, we found that induction of IL-6 mRNA occurred very early on reperfusion of the infarcted myocardium. We hypothesized that the release of a preformed upstream cytokine induced IL-6 in leukocytes infiltrating on reperfusion. METHODS AND RESULTS: Constitutive expression of TNF-alpha and not IL-1beta was demonstrated in the normal canine myocardium and was localized predominantly in cardiac mast cells. Mast cell degranulation in the ischemic myocardium was documented by demonstration of a rapid release of histamine and TNF-alpha in the cardiac lymph after myocardial ischemia. Histochemical studies with FITC-labeled avidin demonstrated degranulating mast cells only in ischemic samples of canine myocardium. Immunohistochemistry suggested that degranulating mast cells were the primary source of TNF-alpha in the ischemic myocardium. In situ hybridization studies of reperfused myocardium localized IL-6 mRNA in infiltrating mononuclear cells and in mononuclear cells appearing in the postischemic cardiac lymph within the first 15 minutes of reperfusion. Furthermore, isolated canine mononuclear cells incubated with postischemic cardiac lymph demonstrated significant induction of IL-6 mRNA, which was partially blocked with a neutralizing antibody to TNF-alpha. CONCLUSIONS: Cardiac mast cells degranulate after myocardial ischemia, releasing preformed mediators, such as histamine and TNF-alpha. We suggest that mast cell-derived TNF-alpha may be a crucial factor in upregulating IL-6 in infiltrating leukocytes and initiating the cytokine cascade responsible for myocyte ICAM-1 induction and subsequent neutrophil-induced injury.

Animals↗
PubMed1998

Spontaneous skin ulceration and defective T cell function in CD18 null mice.

A null mutation was prepared in the mouse for CD18, the beta2 subunit of leukocyte integrins. Homozygous CD18 null mice develop chronic dermatitis with extensive facial and submandibular erosions. The phenotype includes elevated neutrophil counts, increased immunoglobulin levels, lymphadenopathy, splenomegaly, and abundant plasma cells in skin, lymph nodes, gut, and kidney. Very few neutrophils were found in spontaneously occurring skin lesions or with an induced toxic dermatitis. Intravital microscopy in CD18 null mice revealed a lack of firm neutrophil attachment to venules in the cremaster muscle in response to N-formyl- methionyl-leucyl-phenylalanine. A severe defect in T cell proliferation was found in the CD18 null mice when T cell receptors were stimulated either by staphylococcal enterotoxin A or by major histocompatibility complex alloantigens demonstrating a greater role of CD11/CD18 integrins in T cell responses than previously documented. The null mice are useful for delineating the functions of CD18 in vivo.

Animals↗
PubMed1998

P-Selectin support of neonatal neutrophil adherence under flow: contribution of L-selectin, LFA-1, and ligand(s) for P-selectin.

To further define the neonatal neutrophil's ability to localize to inflamed tissue compared with adult cells, we examined the neonatal neutrophil interactions with P-selectin monolayers under two conditions: (1) attachment under constant shear stress and flow and (2) detachment where cells were allowed to attach in the absence of shear stress and then shear stress is introduced and increased in step-wise increments. Cord blood and adult neutrophils had minimal interactions with unstimulated human umbilical vein endothelial cells (HUVECs) at a constant shear stress of 2 dynes/cm2. There was a marked increase in the number of both neonatal and adult cells interacting (interacting cells = rolling + arresting) with HUVECs after histamine stimulation, although the neonatal value was only 40% of adult (P < .05). Neonatal neutrophils also had significantly decreased interaction with monolayers of Chinese hamster ovary (CHO) cells transfected with human P-selectin (CHO-P-selectin; 60% of adult values, P < .003). Of the interacting cells, there was a lower fraction of neonatal cells that rolled compared with adult cells on both stimulated HUVECs and CHO-P-selectin. That neonatal neutrophil L-selectin contributes to the diminished attachment to P-selectin is supported by the following: (1) Neonatal neutrophils had significantly diminished expression of L-selectin. (2) Anti-L-selectin monoclonal antibody reduced the number of interacting adult neutrophils to the level seen with untreated neonatal neutrophils, but had no effect on neonatal neutrophils. In contrast, L-selectin appeared to play no role in maintaining the interaction of either neonatal or adult neutrophils in the detachment assay. Once attachment occurred, the neonatal neutrophil's interaction with the P-selectin monolayer was dependent on LFA-1 and to other ligands to a lesser degree based on the following: (1) Control neonatal neutrophils had decreased rolling fraction compared with adult neutrophils, although the total number of interacting neutrophils was equal between groups. (2) Anti-LFA-1 treatment resulted in an increase in the rolling fraction of both neonatal and adult neutrophils. However, whereas the number of interacting adult neutrophils remained unchanged, the number of neonatal neutrophils decreased with increased shear stress. We speculate that this increased detachment of neonatal cells is due to differences in neutrophil ligand(s) for P-selectin.

Adult↗
PubMed1998

Beta2-integrins mediate stable adhesion in collisional interactions between neutrophils and ICAM-1-expressing cells.

The aggregation of human neutrophils in suspension has features that are analogous to their attachment to activated endothelium in that both involve selectin and beta2-integrin adhesion receptors. For the collisional interaction that forms neutrophil aggregates in suspension, there is a tethering step in which L-selectin on neutrophils binds PSGL-1. At relatively low shear rates (100-200 s(-1)) firm adhesion is mediated in equal measure by LFA-1 binding to ICAM-3, and Mac-1 binding to an as yet undefined ligand. In this report we used a mouse melanoma cell line expressing an estimated 700,000 ICAM-1 (CD54) to examine the relative roles of LFA-1 and Mac-1 over the kinetics of heterotypic cell adhesion in shear mixed suspensions. Neither heterotypic nor homotypic neutrophil aggregates formed with application of shear alone. However, the rate of aggregation peaked within seconds of chemotactic stimulation. In contrast to homotypic aggregation, neither L-selectin nor its O-glycoprotein ligands on neutrophils contributed to heterotypic adhesion. Adhesion was inhibited in a dose-dependent manner as ICAM-1 was titrated with blocking mAb. A direct interaction between LFA-1 and ICAM-1 was preferred over the first minute of stimulation, whereas at later times adhesion was supported equally by Mac-1. Activation with MnCl2 also favored participation of the constitutively expressed LFA-1. Application of defined shear in a cone and plate viscometer showed that adhesion to the ICAM-1 cells decreased from a maximum level to baseline as shear rate increased up to 400 s(-1) in a manner typical of integrin adhesion alone. In contrast, homotypic aggregation supported by the transition from selectin to integrin binding exhibited an increase in efficiency up to 800 s(-1). The pathophysiological significance of receptor site density and duration of contact in collisional interactions relevant to leukocyte recruitment compared to leukocyte-endothelial cell interactions on surfaces is discussed.

Animals↗