Bonnie J. Hughes

Recognition of an Endothelial Determinant for CD18-dependent Human Neutrophil Adherence and Transendothelial Migration

Human neutrophil (PMN) attachment to human umbilical vein endothelial cells (HUVEC) was evaluated in vitro using two MAbs, R6-5-D6 and RR1/1, that recognize intercellular adhesion molecule-1 (ICAM-1), and one MAb, TS1/18, that recognizes CD18. Pretreatment of the HUVEC with anti-ICAM-1 MAbs produced greater than 50% inhibition of attachment to HUVEC, and IL-1 (0.5 U/ml)- or lipopolysaccharide (LPS) (10 ng/ml)-stimulated HUVEC, and greater than 99% inhibition of f-Met-Leu-Phe (0.5 nM) enhanced adherence. Anti-ICAM-1 MAbs also inhibited by greater than 85% the transendothelial migration induced by 4-h IL-1 (0.5 U/ml) and LPS (10 ng/ml) activation of the HUVEC. That these effects involved a CD18-dependent mechanism is supported by the following results: pretreatment of PMN with TS1/18 produced the same degree of inhibition of attachment and migration as seen with R6-5-D6. In addition, the use of both MAbs together did not further increase the inhibition of cell attachment to stimulated HUVEC. The attachment of PMN from patients with CD18 deficiency to stimulated HUVEC was not reduced by R6-5-D6, and both R6-5-D6 and TS1/18 revealed the same time course for appearance and disappearance of an adherence component on stimulated HUVEC not blocked by either MAb. These results demonstrate that attachment and transendothelial migration of PMN in vitro depend substantially on both CD18 on the PMN and ICAM-1 on the endothelial cell.

Abnormalities of polymorphonuclear leukocyte function associated with a heritable deficiency of high molecular weight surface glycoproteins (GP138): common relationship to diminished cell adherence.

Investigations of polymorphonuclear leukocyte (PMN) function were performed in a 5-yr-old white female with delayed umbilical cord separation, impaired pus formation, and a severe defect of PMN chemotaxis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated an almost total deficiency of a high molecular weight glycoprotein(s) (GP138) in the granule and membrane fractions of the patients cells, and NaB3H4-galactose oxidase labeling demonstrated the absence of a major glycoprotein complex on the surface of her PMNs. Monoclonal antibodies (MAb) were employed in flow cytometry experiments to demonstrate that two previously characterized glycoproteins (Mo1 and LFA1) were undetectable on the surface of the patients PMNs and monocytes. Immunoprecipitation of 125I-labeled patient cells with subunit specific MAbs confirmed that the alpha-subunits of Mo1 (155 kD) and LFA1 (177 kD) and their common beta-subunit (94 kD) were totally deficient. Functional analyses of patient PMNs demonstrated severe impairment of adherence- and adhesion-dependent cell functions including spreading, aggregation, orientation in chemotactic gradients, antibody-dependent cellular cytotoxicity, and phagocytosis of particles (Oil-Red-0-paraffin, zymosan) selectively opsonized with C3-derived ligands. Patient PMNs demonstrated a normal capacity to rosette with IgG or C3b-coated sheep erythrocytes, but rosette formation with C3bi-coated erythrocytes was profoundly diminished. Adhesion-independent functions including shape change, N-formyl-methionyl-leucyl-3H-phenylalanine binding, and O-2 generation or secretion elicited by soluble stimuli were normal. Membrane fluidity, surface charge, and microtubule assembly were also normal. These findings provide new evidence that critical PMN surface glycoproteins are required to facilitate multiple adhesion-dependent cellular functions of the inflammatory response.

Interleukin-8 gene induction in the myocardium after ischemia and reperfusion in vivo.

Neutrophil adhesion and direct cytotoxicity for cardiac myocytes require chemotactic stimulation and are dependent upon CD18-ICAM-1 binding. To characterize the potential role of IL-8 in this interaction, canine IL-8 cDNA was cloned and the mature recombinant protein expressed in Escherichia coli BL21 cells. Recombinant canine IL-8 markedly increased adhesion of neutrophils to isolated canine cardiac myocytes. This adhesion resulted in direct cytotoxicity for cardiac myocytes. Both processes were specifically blocked by antibodies directed against CD18 and IL-8. In vivo, after 1 h of coronary occlusion, IL-8 mRNA was markedly and consistently induced in reperfused segments of myocardium. IL-8 mRNA was not induced in control (normally perfused) myocardial segments. Minimal amounts of IL-8 mRNA were detected after 3 or 4 h of ischemia without reperfusion. Highest levels of induction were evident in the most ischemic myocardial segments. IL-8 mRNA peaked in the first 3 h of reperfusion and persisted at high levels beyond 24 h. IL-8 staining was present in the inflammatory infiltrate near the border between necrotic and viable myocardium, as well as in small veins in the same area. These findings provide the first direct evidence for regulation of IL-8 in ischemic and reperfused canine myocardium and support the hypothesis that IL-8 participates in neutrophil-mediated myocardial injury.

Recruitment of CD11b/CD18 to the neutrophil surface and adherence-dependent cell locomotion.

Chemotactic stimulation of neutrophils results in translocation of CD11b/CD18 (Mac-1) from intracellular storage pools to the cell surface. Though results from several laboratories indicate that the newly arrived surface Mac-1 is not involved in the adherence induced by the initial stimulus, the present study addresses the hypothesis that this Mac-1 plays a role in subsequent adherence-dependent functions. The response of human neutrophils to changing concentrations of a chemotactic stimulus was evaluated by determining the amount of newly arrived surface Mac-1, and Mac-1-dependent adhesion and locomotion. Small step-wise increases in the concentration of f-Met-Leu-Phe (FMLP) resulted in proportional stepwise increases in surface Mac-1 that plateaued within 2-4 min. This newly arrived Mac-1 supported adhesion to protein-coated surfaces only when the cells were exposed to an additional increase in the FMLP stimulus level. Adherence-dependent cellular locomotion was evaluated in chambers that allowed rapid changes in the stimulus concentration. Repeated small increments in the stimulus level at 200-s intervals resulted in significantly longer migration paths than a single-step increase in the stimulus. The results support the hypothesis that small increments in the chemotactic stimulus bring Mac-1 to the cell surface, and this newly mobilized Mac-1 is available for adherence-dependent locomotion with subsequent increases in the concentration of the stimulus.

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.

Impaired motility of neonatal PMN leukocytes: relationship to abnormalities of cell orientation and assembly of microtubules in chemotactic gradients.

To allow a further understanding of the pathogenesis of impaired stimulated locomotion by polymorphonuclear leukocytes (PMNs) in human neonates, we studied cellular orientation by neonatal PMNs in response to well‐defined chemotactic gradients (Zigmond orientation chambers) and characterized the cytoplasmic microtubule (MT) complex of neonatal PMNs during cell orientation and movement. PMN suspensions obtained from 52 neonates demonstrated a diminished capacity to undergo orientation at all time intervals after exposure to gradients of N‐formyl‐methionyl‐leucyl phenylalanine (f‐Met‐Leu‐Phe) or C5a. Among responding (orienting) neonatal PMNs observed, only 70% (f‐Met‐Leu‐Phe) or 59% (C5a) oriented accurately (toward chemotactic gradients) as compared to values of 96% (f‐Met‐Leu‐Phe) or 92% (C5a) for adult controls. Furthermore, neonatal PMNs failed to alter their direction of orientation/migration when chemotactic gradients were reversed. Similar abnormalities were observed when 10‐fold gradients of f‐Met‐Leu‐Phe were employed over a concentration range between 10−7 and 10−11 M. Employing tubulin immunofluorescence, the cytoplasmic MT complex of‐neonatal PMNs was assessed prior to and after cell exposure to uniform concentrations or gradients of chemotactic factors (CFs). MT assembly by neonatal PMNs studied under these experimental conditions was significantly diminished. Neonatal cell suspensions demonstrated 26 ± 5 (f‐Met‐Leu‐Phe) or 27 ± 6 (C5a) MT/cell as compared to respective values of 36 ± 6 or 35 ± 5 for adult suspensions (P <.001). MT lengths of neonatal PMNs increased from 6.7 ± 1 µm (PBS) to 7.5 ± 1 µm (f‐Met‐Leu‐Phe) or 7.3 ± 1 µm (C5a) as

Impaired Chemotaxigenesis by Type III Group B Streptococci in Neonatal Sera: Relationship to Diminished Concentration of Specific Anticapsular Antibody and Abnormalities of Serum Complement

Summary: A chemotaxigenesis (CTG) assay employing adult or neonatal sera, type III group B streptococci (GBS) and polymorphonuclear leukocytes (PMNs) was designed to evaluate the role of PMN mobilization in the pathogenesis of type III GBS infection in neonates. Generation of C5a in healthy adult sera with moderate-high (3–40 μg/ml) or low (≤2 μg/ml) levels of specific anticapsular antibody was confirmed by PMN aggregometry and by the neutralization of CTG by goat anti-human C5. CTG was significantly (P < 0.001) greater in high as compared to low specific antibody-containing adult sera; stepwise increases in CTG occurred when specific IgG was added to untreated, but not heat-inactivated, hypogammaglobutinemic serum. Immunospecificity of CTG was shown by a failure of type III GBS to generate C5a in heterologous (type Ia) high antibody sera. Mean CTG values in three high and 16 low antibody-containing sera from healthy term neonates were 24% and 62% of high (P < 0.001) and low (P < 0.01) antibody adult sera, respectively. The addition of both complement and specific IgG to low antibody-containing neonatal sera was required to enhance their CTG activity to high antibody adult values. CTG by type III GBS in neonatal sera-neonatal PMN mixtures was only 25% (high antibody sera) and 14% (low antibody sera) of values for paired maternal sera mixtures reacted with adult PMNs (P < 0.001). These studies demonstrate that CTG by type III GBS in neonatal sera is markedly diminished and that low concentrations of specific anticapsular antibody and abnormalities of complement function contribute to impaired PMN mobilization in human neonates.

Clinical and Immunological Features Associated with Bovine Leukocyte Adhesion Deficiency

Granulocytopathies in dogs (1), humans (2), and cattle (3,4) characterized by persistent progressive neutrophilia in patients affected with severe recurrent bacterial infections and failure to form pus were reported between 1975 and 1987. In all three species, these conditions were determined to be heritable deficiencies of leukocyte surface glycoproteins associated with diminished cell adherence between 1984 and 1990 (5–9). Although published reports of its diagnosis are few, the bovine granulocy-topathy syndrome has been diagnosed at veterinary schools throughout the world during the past 8 y. In vitro assessments have identified abnormalities of motile, phagocytic, and oxidative functions of neutrophils which appear to mediate inflammatory deficits in vivo (3,4,7,8). Factors contributing to low frequency of diagnosis of this syndrome may relate to the impracticality of intensive clinical laboratory studies in food-producing animal species.

880 SECRETORY DETERMINANTS OF IMPAIRED ADHERENCE MOTIUTY OF NEONATAL PMNS

To evaluate possible secretory determinants of pathologic neonatal PMN (NP) adherence and stimulated migration, correlative studies of 2° granule lactoferrin (LF) release (RIA), chemotactic factor receptor “up regulation” (fML3HP binding, 4°C), and the induction of OKM1, & p150,95 glycoprotein (GP) surface expression (Flow Cytometry) mediated by secretory or chemotactic stimuli were performed. LF content of 31 healthy term NP suspensions (x 15.2 g/107 PMNs) was diminished (p<.001) compared to that of 38 healthy adult (AP) suspensions (x 31 g/107 PMNs). NP demonstrated diminished (p<.01) LF release in suspension in response to PMA (500 ng/ml) or A23187 (>10−7M) and during adherence to glass substrates in the presence of PMA or fMLP (p<.01). “Up regulation” of specific fML3HP binding (stimulated - baseline values) of NP by PMA (>500 ng/ml) or A23187 (>2.5×10−8M) was also diminished (× CPM×103/107 PMN: PMA; 5.8 (NP), 10.4 (AP), A23187; 9.1 (NP), 21.2 (AP) (p<.001). PMA mediated a minimal enhancement of surface expression of OKM1 α, β, & p150,95α (x fold increase; 1.4, 1.6, 1.5) compared to AP (x fold increase; 5.6, 5.9, 5.3). Diminished (p<.001) induction of these GPs on NP by fMLP or C5a was directly related to impaired enhancement of adherence by these chemotactic factors (r=.89; p<.001). These studies suggest that impaired hyperadherence and stimulated migration by NP are functionally linked to abnormalitles of 2° granules & a resultant diminished availability of LF, fMLP receptors & “adhesive” GPs which are required at the cell surface for these events.

Mac-1 (CD11b/CD18) and Adherence-Dependent Neutrophil Locomotion

CD18 integrins are necessary for adherence-dependent neutrophil locomotion in vitro as shown by the fact that neutrophils from patients with CD18 deficiency exhibit markedly reduced migration on protein coated glass or plastic (1), and on monolayers of endothelial cells (2,3). Adhesion to the these surfaces apparently requires stimulation of the neutrophil (e.g., with chemotactic factors), and involves CD11b/CD18 (Mac-1) for the attachment to protein-coated surfaces (4–7), and both Mac-l and CD11a/CD18 (LFA-1) for attachment to endothelial cells (3). A well known effect of chemotactic stimulation is the mobilization of Mac-1 from secondary granules in the neutrophil to the cell surface (4,8,9–14). The functional significance of this event is uncertain, and recent evidence suggests that constitutively expressed Mac-1 is sufficient for the increased adhesion induced by a single chemotactic stimulus (15–19). In this report, we propose the hypothesis that the newly arrived Mac-1 can participate in adhesion, but our evidence indicates that in order to do so, the neutrophil must experience a further increase in the concentration of the chemotactic stimulus.