Cell adhesion molecules--update

Cell adhesion molecules are glycoproteins expressed on the cell surface and play an important role in inflammatory as well as neoplastic diseases. There are four main groups: the integrin family, the immunoglobulin superfamily, selectins, and cadherins. The integrin family has eight subfamilies, designated as beta 1 through beta 8. The most widely studied subfamilies are beta 1 (CD29, very late activation [VLA] members), beta 2 (leukocyte integrins such as CD11a/CD18, CD11b/CD18, CD11c/CD18, and alpha d beta 2), beta 3 (CD61, cytoadhesions), and beta 7 (alpha 4 beta 7 and alpha E beta 7). The immunoglobulin superfamily includes leukocyte function antigen-2 (LFA-2 or CD2), leukocyte function antigen-3 (LFA-3 or CD58), intercellular adhesion molecules (ICAMs), vascular adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule-1 (PE-CAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The selectin family includes E-selectin (CD62E), P-selectin (CD62P), and L-selectin (CD62L). Cadherins are major cell-cell adhesion molecules and include epithelial (E), placental (P), and neural (N) subclasses. The binding sites (ligands/receptors) are different for each of these cell adhesion molecules (e.g., ICAM binds to CD11/CD18; VCAM-1 binds to VLA-4). The specific cell adhesion molecules and their ligands that may be involved in pathologic conditions and potential therapeutic strategies by modulating the expression of these molecules will be discussed.     

Local anesthetic lidocaine inhibits the effect of granulocyte colony-stimulating factor on human neutrophil functions

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced superoxide (O2-) release in human neutrophils stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and inversely regulated the surface expression of cellular adhesion molecules, leukocyte adhesion molecule-1 (LAM-1) and CD11b/CD18 leukocyte integrin, on human neutrophils; that is, rhG-CSF downregulated the expression of LAM-1 and upregulated the expression of CD11b on neutrophils. The cationic local anesthetic lidocaine inhibited not only FMLP-induced O2- release in neutrophils but also FMLP-induced CD11b upregulation and LAM-1 downregulation on neutrophils in a dose-dependent manner. Lidocaine also abolished the priming effect of rhG-CSF for enhanced release of O2- in neutrophils and inhibited rhG-CSF-induced CD11b upregulation and LAM-1 downregulation on neutrophils in a dose-dependent manner. These findings suggest that lidocaine inhibits human neutrophil functions, such as adherence to endothelial cells, by interfering with the expression of cellular adhesion molecules on neutrophils, and that lidocaine might have anti-inflammatory properties by inhibiting the effect of inflammatory cytokines.     

Are circulating neutrophils intravascularly activated in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides?

Vascular injury in vasculitis may be due to activation of circulating neutrophils resulting in their increased adhesiveness to locally activated endothelium (Shwartzman phenomenon). Previously, we demonstrated up-regulation of endothelial intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in biopsies from patients with ANCA-associated vasculitis. In the present study, we investigated the expression of adhesion molecules (CD11b, ICAM-1, VLA-4, L-selectin) and activation markers (CD66b, CD64, CD63) on circulating neutrophils from patients with ANCA-associated vasculitis in comparison with their expression on cells from healthy volunteers and patients with sepsis. We related these findings to parameters of disease activity. Surface marker expression was determined by using a non-activating whole blood flow cytometric assay. The expression of activation markers, but not the expression of adhesion molecules, was increased on neutrophils from patients with active vasculitis. The expression of CD63 and CD66b on neutrophils correlated with disease activity as determined by the Birmingham Vasculitis Activity Score (BVAS). In contrast to patients with active vasculitis, patients with sepsis showed up-regulation of all markers, including adhesion molecules, suggesting that circulating neutrophils are fully activated in sepsis. We conclude that in ANCA-associated vasculitis, circulating neutrophils are not fully activated, since they do not express increased levels of adhesion molecules as sepsis or in the Shwartzman reaction. These findings are compatible with the concept that in vivo vascular damage in ANCA-associated vasculitides does not occur due to a Shwarzman-like reaction but only after ANCA-induced neutrophil activation at the endothelial cell surface.     

Effect of theophylline on CD11b and L-selectin expression and density of eosinophils and neutrophils in vitro

The nonspecific phosphodiesterase inhibitor theophylline, widely used in asthma therapy, may cause a decrease in inflammatory responses of airways. In asthma, eosinophils migrate to the airway wall and become activated. Activated eosinophils are characterized by low cell density, as well as increased expression of CD11b and reduced expression of L-selectin, two adhesion molecules involved in transendothelial migration. To study the anti-inflammatory effect of theophylline on granulocyte adhesion molecules in vitro, the platelet-activating factor (PAF)-induced density shift was determined by density centrifugation and the modulation of CD11b and L-selectin expression by flow cytometry on eosinophils and neutrophils in human whole blood. A relatively high concentration of theophylline (10(-3) M) inhibited the increase in the percentage of hypodense eosinophils and neutrophils in whole-blood samples after PAF stimulation in vitro. A more pharmacological concentration (10(-4) M) inhibited the CD11b upregulation and L-selectin shedding induced by PAF (10(-7) M) on both eosinophils and neutrophils. The effect of isoproterenol on the inhibitory effect of theophylline was mainly additive, but a small synergistic effect could not be excluded. In conclusion theophylline can attenuate eosinophil and neutrophil activation in vitro at the level of adhesion molecule expression and changes in cell density. This may have implications for transendothelial migration of these cells in asthma.     

Leukocyte mobilization to skin lesions, determination of cell surface receptors (CD11b/CD18) and phagocytic capacities of neutrophils in dogs with chronic deep pyoderma

A suction blister technique was used in eight dogs with chronic deep pyoderma to determine chemotaxis in vivo. By flow cytometry the expression of adhesion molecules (CD11b/CD18) on exudative and peripheral neutrophils were analyzed in 11 healthy dogs and six dogs with chronic deep pyoderma. Phagocytosis in vitro capacities of exudative and peripheral neutrophils were analyzed in six healthy dogs and six dogs with chronic deep pyoderma. Dogs with chronic pyoderma showed significantly better chemotaxis in vivo compared with the healthy dogs (P < 0.05). Expression of adhesion molecules CD11b and CD18, and phagocytosis was significantly (P < 0.05) better in the dogs with pyoderma compared with the healthy dogs. In both groups exudative cells expressed significantly (P < 0.05) more CD11b/CD18 receptors compared with blood neutrophils. We conclude that there are no serious functional disturbances detectable in the peripheral neutrophils, nor in the exudative neutrophils from dogs with chronic deep pyoderma.     

Adhesion and cytosolic dye transfer between macrophages and intestinal epithelial cells

Activated macrophages (M phi) found in the intestinal lesions of patients with inflammatory bowel disease (IBD) secrete many inflammatory mediators which can regulate intestinal epithelial cell (IEC) function. However, little is known about direct M phi-IEC interactions. Two potential mechanisms by which cells may interact are through specific receptor-ligand binding of adhesion molecules, such as integrins or cadherins, and by exchange of cytoplasmic substances through transmembraneous channels called gap junctions. We investigated whether M phi could adhere to epithelial cells in culture and form transmembrane communication channels as defined by dye transfer. Primary cultures of murine M phi and a M phi cell line, P388D1, adhered to Mode-K and IEC6, but not CMT-93 IEC. Antibody blocking studies determined that P388D1-Mode-K binding was partially dependent on beta 2 integrin (CD18) function, Mode-K constitutively expressed CD106 (VCAM-1) and cell associated fibronectin, while P388D1 expressed low levels of CD49d/CD29 (VLA4) but blocking antibodies to these surface molecules did not inhibit P388D1-Mode-K adherence. Transfer of calcein dye from M phi to IEC was quantitated by flow cytometry and was dependent on M phi-IEC adhesion. Dye transfer was concentration dependent in that the fluorescence intensity of Mode-K was proportional to the number of adherent P388D1 cells as well as the dye load of the M phi. These results indicate that M phi interact with IEC by adhesion and possibly through gap junctions and may thus regulate IEC function by direct cell-cell communication.     

Increased expression of CD11b/CD18 on phagocytes in ischaemic disease: a bridge between inflammation and coagulation

The aim of this study was to assess the expression of CD11b/CD18 integrin adhesion molecules on the phagocytes of patients with ischaemic diseases, and to evaluate the concentration of soluble adhesion molecules that are released from endothelium (sICAM-1) and from phagocytes (sL-selectin). A total of 370 patients were enrolled: 120 with coronary artery disease (CAD); 50 with peripheral artery occlusive disease (PAOD); and 200 control subjects with no clinical manifestations of ischaemic disease. CD11b/CD18 integrin was detected by flow cytometry, whereas sL-selectin and sICAM-1 concentrations were detected using a sandwich-type immunoassay. CD11b/CD18 integrin expression was found to be higher in the patients with ischaemic disease than in the control subjects (P < 0.001). The PAOD patients had higher values of CD11b/CD18 integrin than the CAD ones (P < 0.01). The concentration of soluble adhesion molecules did not show any significant differences within the three groups (P = NS). The high expression of CD11b/CD18 integrin in ischaemic disease patients may depend on the increased, but probably stable, cytokine network that has been demonstrated to occur in chronic ischaemic diseases: the difference observed between PAOD and CAD patients could be the consequence of higher inflammatory activation probably resulting from the greater extent of the atherosclerotic process in PAOD, or of the more localized ischaemic area in CAD patients. CD11b/CD18 can therefore be considered a marker of chronic phagocyte activation during ischaemic disease. On the other hand, sICAM and sL-selectin concentrations were found to be within the normal range; they have recently been considered as a marker for acute ischaemic events and acute inflammatory process activation. Our results confirm that in uncomplicated atherosclerosis no acute inflammatory process activation should occur.     

Ionizing radiation mediates expression of cell adhesion molecules in distinct histological patterns within the lung

Inflammatory cell infiltration of the lung is a predominant histopathological change that occurs during radiation pneumonitis. Emigration of inflammatory cells from the circulation requires the interaction between cell adhesion molecules on the vascular endothelium and molecules on the surface of leukocytes. We studied the immunohistochemical pattern of expression of cell adhesion molecules in lungs from mice treated with thoracic irradiation. After X-irradiation, the endothelial leukocyte adhesion molecule 1 (ELAM-1; E-selectin) was primarily expressed in the pulmonary endothelium of larger vessels and minimally in the microvascular endothelium. Conversely, the intercellular adhesion molecule 1 (ICAM-1; CD54) was expressed in the pulmonary capillary endothelium and minimally in the endothelium of larger vessels. Radiation-mediated E-selectin expression was first observed at 6 h, whereas ICAM-1 expression initially increased at 24 h after irradiation. ICAM-1 and E-selectin expression persisted for several days. P-selectin is constitutively expressed in Weibel-Palade bodies in the endothelium, which moved to the vascular lumen within 30 min after irradiation. P-selectin was not detected in the pulmonary endothelium at 6 h after irradiation. The radiation dose required for increased cell adhesion molecule expression within the pulmonary vascular endothelium was 2 Gy, and expression increased in a dose-dependent manner. These data demonstrate that ICAM-1 and E-selectin expression is increased in the pulmonary endothelium following thoracic irradiation. The pattern of expression of E-selectin, P-selectin, and ICAM-1 is distinct from one another.     

Expression of cell adhesion molecules in placentae from pregnancies complicated by pre-eclampsia and intrauterine growth retardation

Platelets and neutrophils are involved in maternal placental vascular damage in pre-eclampsia. Recruitment of these cells is probably mediated by cell adhesion molecules expressed at the uteroplacental bed. It remains controversial as to whether platelets and neutrophils mediate damage to trophoblast or villous vasculature. The purpose of this study was to determine the expression of cell adhesion molecules in placentae from normal pregnancies and pregnancies complicated by pre-eclampsia and intrauterine growth retardation (IUGR). Immunostaining for platelet endothelial cell adhesion molecule (PECAM) and intercellular adhesion molecule-1 (ICAM-1) was localized mainly to the endothelium of stem villi, intermediate villi, terminal villi and decidual vessels. Scattered staining for ICAM-1 was also evident in the stroma and fetal membranes. The endothelium of stem villi, intermediate villi and terminal villi were all negative for vascular cell adhesion molecule-1 (VCAM-1) and E-Selectin. PECAM, ICAM-1 and ICAM-2 mRNA were all detectable in normal placentae using northern blotting analysis whereas mRNA for E-Selectin and VCAM-1 were both undetectable. There were no differences in cell adhesion molecule immunostaining or mRNA expression in placentae from pregnancies complicated by pre-eclampsia and IUGR inconclusion, expression of cell adhesion molecules in placentae from pre-eclampsia and IUGR are consistent with a normal physiological role in vascular function.     

Alprazolam 0.125 mg twice a day improves aspects of psychometric performance in healthy volunteers

METHODS: To examine the inflammatory process in chronic bronchitis, we evaluated the cell and cytokine profile in bronchoalveolar lavage fluid from 12 chronic bronchitis patients who smoked, six chronic bronchitis patients who did not smoke, 10 subjects control subjects without pulmonary diseases who smoked and eight control subjects who did not smoke. RESULTS: Chronic bronchitis patients who smoked had increased numbers of macrophages, neutrophils, eosinophils, mast cells and activated CD8+ T lymphocytes and predominantly expressed interleukin-8, tumour necrosis factor alpha and interleukin-2 genes and proteins. The number of macrophages and neutrophils and the expression of interleukin 8 were also increased in control subjects who smoked compared with healthy subjects who did not smoke. Chronic bronchitis patients who did not smoke had increased numbers of eosinophils, mast cells and activated CD4+ T lymphocytes and predominantly expressed interleukin-5 and granulocyte-macrophage colony-stimulating factor genes and proteins. CONCLUSION: Thus, the cellular and molecular characteristics of the inflammatory process in chronic bronchitis patients who smoke and do not smoke are different, suggesting a different pathogenesis of the disease.     

Functional association of platelet endothelial cell adhesion molecule-1 and phosphoinositide 3-kinase in human neutrophils

In this paper we show that the engagement of the platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) up-regulates the adhesion of human neutrophils to the EA.hy926 endothelial cell line through a phosphoinositide 3-kinase (PI3K)-dependent pathway. Indeed, LY294002 and wortmannin prevented the effect of PECAM-1/CD31 cross-linking on cell adhesion, at concentrations known to inhibit PI3K without affecting other kinases. Both compounds blocked neutrophil binding to murine fibroblasts transfected with human ICAM-1, to purified ICAM-1 protein, or to fibronectin, suggesting that PECAM-1/CD31-mediated up-regulation of beta2 and beta1 integrin-mediated adhesion is PI3K-sensitive. We also provide evidence for the association of PECAM-1/CD31 to PI3K, because PI3K was detectable in neutrophil lysates after PECAM-1/CD31 cross-linking and immunoprecipitation. PECAM-1/CD31-dependent recruitment of PI3K was suggested by the finding that the serine/threonine kinase p70 S6 kinase (S6K), a signaling protein downstream of PI3K, is activated in neutrophils upon PECAM-1/CD31 cross-linking, based on the appearance of serine phosphorylation in S6K immunoprecipitates. In turn, S6K is not directly involved in the up-regulation of integrin function because rapamycin, which can inhibit S6K independent of PI3K, did not block PECAM-1/CD31-induced adhesion of neutrophils to beta1 and beta2 integrin substrates. In conclusion, PECAM-1/CD31 appears to be one of the molecules functionally coupled to PI3K, suggesting that this enzyme may represent a common pathway of integrin and adhesiveness regulation in leukocytes.     

Leukocyte adhesion molecules in the liver and plasma cytokine levels in endotoxin-induced rat liver injury

BACKGROUND: The interactions between polymorphonuclear neutrophils (PMNs) and sinusoidal endothelial cells (SECs) have been known to be involved in the pathogenesis of acute liver injury. It has been also reported that tumor necrosis factor-alpha (TNF-alpha) up-regulates ICAM-1 expression on SECs and that interleukin-8 (IL-8) provokes rapid activation of CD11/CD18 on PMNs. These findings expand into the relationship between the expression of leukocyte adhesion molecules (ICAM-1, CD11a/CD18 and CD11b/CD18) in liver tissues and plasma TNF and IL-8 levels after lipopolysaccharide (LPS)-induced liver injury in rats. METHODS: Male Wistar rats weighing 200-250 g were treated with 2 mg LPS/kg intravenously in a 0.2- to 0.25-ml volume. Liver and blood samples were obtained at 1, 3, 8, and 12 h after LPS exposure. Plasma TNF and IL-8 levels were measured using bioassay and specific enzyme-linked immunosorbent assay, respectively. Liver samples were fixed and studied by immunohistochemistry using specific monoclonal antibodies against ICAM-1, CD11a, and CD11b. RESULTS: The TNF level showed a peak at 1 h (23.3 +/- 11.4 IU/ml), and the IL-8 level showed a peak at 3 h (343.1 +/- 110.5 ng/ml) after LPS exposure. An increase in the number of PMNs in the liver was observed as early as 1 h and continued until 12 h after LPS exposure. PMNs adhered to degenerated SECs and hepatocytes. ICAM-1 on SECs was diffusely and strongly expressed at 8 h, and PMNs adhered to SECs expressed both CD11a and CD11b. ICAM-1 was also observed on hepatocytes. CONCLUSION: These data suggest that PMN-SEC and PMN-hepatocyte interactions via leukocyte adhesion molecules, related to inflammatory cytokines such as TNF and IL-8, exist and play an important role in the pathogenesis of acute liver injury.     

The protein tyrosine kinase p56lck regulates cell adhesion mediated by CD4 and major histocompatibility complex class II proteins

The CD4 protein is expressed on a subset of human T lymphocytes that recognize antigen in the context of major histocompatibility complex (MHC) class II molecules. Using Chinese hamster ovary (CHO) cells expressing human CD4, we have previously demonstrated that the CD4 protein can mediate cell adhesion by direct interaction with MHC class II molecules. In T lymphocytes, CD4 can also function as a signaling molecule, presumably through its intracellular association with p56lck, a member of the src family of protein tyrosine kinases. In the present report, we show that p56lck can affect cell adhesion mediated by CD4 and MHC class II molecules. The expression of wild-type p56lck in CHO-CD4 cells augments the binding of MHC class II+ B cells, whereas the expression of a mutant p56lck protein with elevated tyrosine kinase activity results in decreased binding of MHC class II+ B cells. Using site-specific mutants of p56lck, we demonstrate that the both the enzymatic activity of p56lck and its association with CD4 are required for this effect on CD4/MHC class II adhesion. Further, the binding of MHC class II+ B cells induces CD4 at the cell surface to become organized into structures resembling adhesions-type junctions. Both wild-type and mutant forms of p56lck influence CD4-mediated adhesion by regulating the formation of these structures. The wild-type lck protein enhances CD4/MHC class II adhesion by augmenting the formation of CD4-associated adherens junctions whereas the elevated tyrosine kinase activity of the mutant p56lck decreases CD4-mediated cell adhesion by preventing the formation of these structures.     

Progression of cochlear and retinal degeneration in the tubby (rd5) mouse

Various molecules expressed on the surface of platelets have been shown to mediate the protective or deleterious role of these cells in immuno-inflammatory mechanisms. Increasing evidence points to the involvement of the cell adhesion molecules, gpIIb-IIIa, P-selectin, CD31, LFA-1, and CD36 in the interaction between platelets and endothelial cells as well as other cell types. The possible role of these molecules in the ability of platelets to support endothelium and to protect against tumour necrosis factor mediated cytolysis or parasitic invasion are reviewed. The involvement of platelets as effectors of tissue damage in cerebral malaria, lipopolysaccharide induced pathology, and pulmonary fibrosis is also discussed. This has then been extended to include the intercellular mechanisms underpinning their pathogenic role in metastasis, transplant rejection, stroke, brain hypoxia, and related conditions. A better understanding of the complex regulation and hierarchical organisation of these various platelet adhesion molecules may prove useful in the development of new approaches to the treatment of such diseases.     

The antigen-presenting cell function of Reed-Sternberg cells

Reed-Sternberg cells, the neoplastic cells of Hodgkin's disease, express all membrane molecules required to function as antigen-presenting cells (APCs), such as major histocompatibility complex (MHC) class II antigens and the recently characterized B7 proteins, which are of critical importance for APC to adequately stimulate CD4+ T cells. As APC do, Reed-Sternberg cells also express the adhesion molecules ICAM-1 (CD54) and LFA-3 (CD58), via which T cells are able to adhere to the cell. MHC antigens, B7 proteins as well as the adhesion molecules are expressed by Reed-Sternberg cells in virtually all cases of Hodgkin's disease, irrespective of the subtype. In vitro studies have shown that Hodgkin's disease-derived cell lines are potent stimulators of mixed lymphocyte cultures and that the MHC antigens, B7 proteins and the adhesion molecules, expressed by Hodgkin's disease-derived cell lines, are essential for such a function. Taken together, these data strongly suggest that Reed-Sternberg cells function as APC in vivo, and that the APC function of the cell is a major common denominator of Hodgkin's disease. The APC function of Reed-Sternberg cells does not support the hypothesis that they derive from dendritic cells, since activated B and T cells may also exert an APC function. Analysis of the antigens that are potentially expressed by Reed-Sternberg cells may greatly advance our knowledge on the pathogenesis of Hodgkin's disease and may allow the development of immunotherapy as an alternative treatment method.     

Late-onset chronic inflammatory encephalopathy in immune-competent and severe combined immune-deficient (SCID) mice with astrocyte-targeted expression of tumor necrosis factor

To examine the role of tumor necrosis factor (TNF)-alpha in the pathogenesis of degenerative disorders of the central nervous system (CNS), transgenic mice were developed in which expression of murine TNF-alpha was targeted to astrocytes using a glial fibrillary acidic protein (GFAP)-TNF-alpha fusion gene. In two independent GFAP-TNFalpha transgenic lines (termed GT-8 or GT-2) adult (>4 months of age) animals developed a progressive ataxia (GT-8) or total paralysis affecting the lower body (GT-2). Symptomatic mice had prominent meningoencephalitis (GT-8) or encephalomyelitis (GT-2) in which large numbers of B cells and CD4+ and CD8+ T cells accumulated at predominantly perivascular sites. The majority of these lymphocytes displayed a memory cell phenotype (CD44high, CD62Llow, CD25-) and expressed an early activation marker (CD69). Parenchymal lesions contained mostly CD45+ high, MHC class II+, and Mac-1+ cells of the macrophage microglial lineage with lower numbers of neutrophils and few CD4+ and CD8+ T cells. Cerebral expression of the cellular adhesion molecules ICAM-1, VCAM-1, and MAdCAM as well as a number of alpha- and beta-chemokines was induced or upregulated and preceded the development of inflammation, suggesting an important signaling role for these molecules in the CNS leukocyte migration. Degenerative changes in the CNS of the GFAP-TNFalpha mice paralleled the development of the inflammatory lesions and included primary and secondary demyelination and neurodegeneration. Disease exacerbation with more extensive inflammatory lesions that contained activated cells of the macrophage/microglial lineage occurred in GFAP-TNFalpha mice with severe combined immune deficiency. Thus, persistent astrocyte expression of murine TNF-alpha in the CNS induces a late-onset chronic inflammatory encephalopathy in which macrophage/microglial cells but not lymphocytes play a central role in mediating injury.     

Emigrated rat neutrophils adhere to cardiac myocytes via alpha 4 integrin

Previous work has shown that neutrophils isolated from whole blood adhere to cardiac-myocytes via CD18 (beta 2 integrin) to cause injury to the heart cells. In vitro, we have found that upon endothelial transmigration, neutrophils can also express alpha 4 beta 1; however, whether this contributes to neutrophil adhesion to parenchymal cells remains entirely unknown. Unstimulated and tumor necrosis factor-alpha-stimulated rat cardiac myocytes adherent to gelatin-coated coverslips supported N-formyl-Met-Leu-Phe (fMLP)-induced neutrophil (isolated from whole blood) adhesion entirely via CD18 (blocked with monoclonal antibody [mAb] WT-3). Emigrated neutrophils spontaneously adhered to cardiac myocytes also entirely via CD18. However, if fMLP was used to restimulate emigrated neutrophils, the adhesion to cardiac myocytes was entirely independent of CD18. Although an anti-alpha 4 integrin antibody (mAb TA-2) alone did not reduce the emigrated neutrophil-myocyte interaction, dual administration of TA-2 and WT-3 reduced adhesion by 81%. alpha 4 integrin was expressed in small amounts on the surface of circulating neutrophils, increased following transmigration, and then increased > 5-fold after restimulation of these emigrated neutrophils. In the presence of the anti-CD18 antibody, a fibronectin fragment (FN-40) but not a vascular cell adhesion molecule-1 antibody (mAb 5F10) inhibitied neutrophil-myocyte interactions by 80%. Similar results were seen when the rat chemokine CINC-gro was used instead of fMLP, suggesting that the alpha 4-dependent adhesion was not specific to fMLP. These data demonstrate that alpha 4 integrin can be physiologically induced to increase in number and avidity after neutrophil emigration and that this adhesion molecule can cause firm adhesion to fibronectin on parenchymal cells, including rat cardiac myocytes.     

Canine neutrophil adhesion proteins and Fc-receptors in healthy dogs and dogs with adhesion protein deficiency, as studied by flow cytometry

Murine monoclonal anti-human antibodies directed against neutrophil adhesion protein receptors CD35, CD18, CD11b, CD11c and the Fc-receptors CD64 (Fc gamma RI), CD32 (FC gamma RII) and CD16 (Fc gamma RIII) were evaluated regarding their ability to bind to the canine homologues. The antibodies against CD35, CD18, CD11b, CD11c and CD16 could be used to evaluate the expression of canine homologues. The routine of using frozen cells and thereby avoiding methodological errors, when samples are stained at different times, was evaluated by comparison of receptor expression in frozen and fresh samples from the same dogs. All receptors were expressed consistently on the cell surface on frozen and fresh neutrophils with the exception of CD16, which showed decreased expression in frozen cells. The expression of CD11c on neutrophils from dogs with canine leucocyte adhesion deficiency (CLAD) was analyzed and there was no difference in receptor expression between CLAD-puppies and healthy controls. CD11b/CD18 expression on neutrophil samples from three parents of CLAD-puppies, i.e. heterozygotes, did not differ from receptor expression in normal controls. Analysis of the Fc-receptor expression on neutrophils from CLAD-puppies showed that the expression of CD16 tended to be decreased in patients compared with controls.     

CD18-independent mechanism of neutrophil emigration in the rabbit lung after ischemia-reperfusion

BACKGROUND: Reperfusion of ischemic lung causes an inflammatory pulmonary vascular injury characterized by increased vascular permeability and migration of inflammatory cells into the alveoli. Migration of neutrophils into the alveolus during reperfusion after 24 hours of unilateral pulmonary artery occlusion has been shown to be in part dependent on the CD18 adhesion molecule on the cell surface. The current study investigated whether reperfusion lung injury after a 1-hour period of complete lung ischemia was CD18 dependent. METHODS: Eighteen rabbits were assigned to one of three groups. Groups 1 and 2 were subjected to one hour of in situ right hilar occlusion followed by 2 hours of reperfusion. Group 3 was subjected to identical surgical dissection but the right hilum was never occluded. Group 1 rabbits received saline solution (1 mL/kg) before hilar occlusion and group 2 rabbits, monoclonal antibody 60.3, a blocking antibody for the CD18 adhesion molecule on the neutrophil surface (2 mg/kg). In 3 of the antibody-treated rabbits, flow cytometry was performed on blood neutrophils before and after administration of the antibody and 120 minutes after reperfusion. RESULTS: The rabbits in groups 1 and 2 had significantly increased alveolar neutrophil infiltrate and increased pulmonary vascular resistance compared with the rabbits in group 3. However, there was no significant difference between group 1 (saline solution treated) and group 2 (antibody treated). Antibody treatment did not block migration of neutrophils into the alveoli. Flow cytometry of circulating neutrophils demonstrated that CD18 was upregulated after reperfusion and that CD18 was fully blocked after antibody treatment for the duration of the study. CONCLUSIONS: We conclude that a 1-hour period of warm ischemia followed by reperfusion results in upregulation of CD18 but that emigration of the neutrophils into the alveoli is not CD18 dependent in this injury.