Effects of trauma and sepsis on soluble L-selectin and cell surface expression of L-selectin and CD11b

OBJECTIVES: To examine (1) the effects of trauma on changes in neutrophil L-selectin and CD11b expression and on the levels of soluble L-selectin and (2) whether these alterations are different on leukocyte subpopulations in those patients who develop multiple organ dysfunction syndrome. MATERIALS AND METHODS: Twenty patients with Injury Severity Score (ISS) > or = 16 and 15 patients with ISS score < 16 were studied. Arterial blood were collected serially after injury. The staining of leukocyte surface adhesion molecules was performed with antibodies against L-selectin and CD11b. Positive cell count and mean fluorescence intensity were determined by flow cytometry. Soluble L-selectin was measured using enzyme-linked immunosorbent assay. RESULTS: In patients with ISS > or = 16, neutrophil L-selectin expression showed an immediate increase, reaching peak levels between 3 to 4 hours after injury (p < 0.05 vs. patients with ISS < 16), followed by a gradual decrease. Plasma levels of soluble L-selectin reached peak levels at 6 hours after injury. However, in patients with ISS < 16, minimal changes in L-selectin expression and soluble L-selectin were observed. Neutrophil CD11b expression showed an immediate increase for the first 3 hours followed by a gradual increase up to 24 hours after injury. In patients who developed multiple organ dysfunction syndrome, CD11b both on neutrophils and lymphocytes remained elevated for 120 hours. CONCLUSIONS: These findings suggest that acute neutrophil activation is an early event after trauma and may be implicated as "a vulnerable window" for leukocyte-mediated end organ injury.     

Microbiology of ligature-induced marginal inflammation around osseointegrated implants and ankylosed teeth in cynomolgus monkeys (Macaca fascicularis)

The aim of this study was to characterize the changes in the quantitative expression of beta 2-integrins and L-selectin detected by means of fluorochrome-conjugated monoclonal antibodies and flow cytometry on leukocytes in the systemic circulation after a major musculoskeletal trauma, i.e. hip replacement surgery, and to relate these changes to parameters of the acute-phase response [plasma acute-phase reactants (C-reactive protein, CRP, and interleukin-6, IL-6) and parameters of coagulation activation (thrombin-antithrombin III complexes, TAT)]. Eight patients with either primary or secondary osteoarthritis of the hip received uncemented total hip prostheses. LFA-1 (CD11a/CD18) was upregulated on granulocytes during the operation. MAC-1 (CD11b/CD18) expression on monocytes increased to peak levels 20 h after surgery, whereas the L-selectin (CD62L) expression on monocytes and granulocytes reached peak values at the end of surgery. The changes in expression of LFA-1 on monocytes, MAC-1 on granulocytes and p150,95 (CD11c/CD18) on monocytes and granulocytes during and after the operation did not reach statistical significance. TAT and IL-6 increased during surgery and reached peak values at the end of the operation and 20 h after surgery, respectively. In contrast, CPR concentrations increased after surgery with peak levels 44 h postoperatively. Significant upregulation of LFA-1 on granulocytes and L-selectin on monocytes and granulocytes preceded the increase in IL-6 which again preceded the increase in CRP. However, the up- or downregulation of leukocyte beta 2-integrins and L-selectin during and after surgery was not significantly correlated with the increase in IL-6. The increases in TAT correlated well with the upregulation of L-selectin on monocytes, but not with the beta 2-integrins known to participate in the coagulation process in vitro. The rise in CRP was inversely correlated with the maximal increase in expression of MAC-1 on monocytes. In conclusion, the changes in leukocyte adhesion molecules during and after surgery indicate changes in critical leukocyte functions. The lack of correlation between quantitative up- and downregulation of leukocyte beta 2-integrins and parameters of the acute phase response suggests that these processes are regulated through independent pathways or that functional up- and downregulation of adhesion molecules, shedding, leukocyte-endothelial adhesion and mobilization of new unactivated cells may result in a net estimate of leukocyte activation not suspected to be positively correlated to acute-phase reactants.     

Effects of monocyte purification and culture on integrin expression

Selective alterations in the surface expression of members of the LeuCAM (leukocyte cell adhesion molecule) family of integrins occur during in vitro culture of human monocytes. Such changes may relate in part to cellular maturation, but also to activation following purification and culture of monocytes. In this paper, we examined the effects of monocyte isolation, adherence during culture and endotoxin exposure on the expression of these molecules and the ligand for LFA-1, ICAM-1 (CD54). Expressions of CD11b, CD18 and CD54, but not CD11a or CD11c, were higher on monocytes freshly isolated by density gradient separation and plastic adherence as compared with cells labelled directly in whole blood. However, the surface expression of the LeuCAMs and CD54 on cultured monocytes was not affected by short-term adherence to plastic for 2 h, as determined by comparisons of their expression on adherence-isolated and elutriated monocytes. In contrast, prolonged adhesion of monocytes for up to 21 days in culture altered expression of CD11a without affecting that of the other LeuCAMs or CD54. Expression of CD11a decreased more rapidly on adherence-maintained cells as compared with suspension-cultured cells. Our results show that cellular manipulations required for in vitro studies of monocyte/macrophages may alter expression of the LeuCAMs.     

The impact of the Dialysis Outcomes Quality Initiative Guidelines on the care of the pediatric end-stage renal disease patient

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.     

Effects of intravenous methylprednisolone therapy on leukocyte and soluble adhesion molecule expression in MS

Intravenous methylprednisolone (IVMP) may inhibit inflammatory cell recruitment to active MS lesions by effects on leukocyte or endothelial cell adhesion molecule expression. We investigated 15 MS patients in relapse receiving a 5-day course of IVMP (500 mg/day) and 15 normal subjects. Patients' blood samples were obtained pretreatment, at 6 and 24 hours after the first dose, and 48 hours after completion of therapy. Levels of L-selectin, leukocyte functional antigen 1 (LFA-1), Mac-1, and very late activation antigen 4 (VLA-4) expression were determined on alphabeta and gammadelta T cells and monocytes by dual-color immunofluorescent flow cytometry. Serum levels of soluble (s) L-selectin, sE-selectin, soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) were measured by ELISA. There was a marked decrease in the T-cell and monocyte counts at 6 hours after therapy, with recovery to baseline at 24 to 48 hours. Adhesion molecule expression was normal on circulating T cells and monocytes in active MS. IVMP resulted in significant changes in the percent adhesion molecule expression on monocytes: increased L-selectin expression at 24 hours, decreased Mac-1 expression at 6 hours, and decreased VLA-4 expression at 6 hours and 24 hours following treatment. T-cell adhesion molecule expression was unaffected by the therapy. Serum sE-selectin was reduced at 6 hours and 24 hours following treatment. IVMP alters the distribution and kinetics of monocyte adhesion molecule expression and endothelial cell release of E-selectin, which may limit monocyte recruitment to areas of tissue destruction in MS.     

Anisotropic expansion in gypsum-bonded cristobalite investment mold

OBJECTIVE: To determine the response of white blood cells in endovascular aortic aneurysm repair. MATERIALS AND METHODS: Seven patients treated with an endoluminal procedure (AAA-E) and seven patients undergoing conventional surgery (AAA-C) were included (all males, aged 52-80 years). A panel of monoclonal antibodies against CD11a, CD11b, CD11c, CD18 and L-selectin was used. To determine the surface receptors on both circulating and sequestered white blood cells, plasma from the patients and cells from healthy donors were combined for flow-cytometry. RESULTS: The expression of CD11a adhesion molecules only showed slight variations regarding granulocytes, but was more pronounced on monocytes, however, without significant differences between the two patient groups, CD11b, CD11c and CD18 molecules on both granulocytes and monocytes were significantly upregulated 60 min after the endovascular procedure compared to conventional aneurysm repair, and L-selectin molecules were by this time correspondingly cleaved off. CONCLUSION: Endovascular aneurysm repair differed significantly from conventional aneurysm surgery with peak adhesion molecule expression 60 min after balloon deflation, probably caused by release of tumour necrosis factor-alpha (TNF-alpha).     

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.     

Sonographic analysis of recurrent parotitis in children: a comparative study with sialographic findings

BACKGROUND: Patients with asthma show altered surface expression of the adhesion molecules CD11b and L-selectin on airway granulocytes compared with blood granulocytes. OBJECTIVE: To investigate whether this modulation is related to disease activity or due to transendothelial migration, we compared the CD11b and L-selectin expression on blood and induced sputum eosinophils and neutrophils between patients with asthma and normal subjects. METHODS: Eleven normal subjects (21-43 years), nine patients (21-34 years) with mild atopic asthma and 10 patients (20-47 years) with moderate to severe atopic asthma on regular treatment with inhaled steroids underwent sputum induction by inhalation of nebulized hypertonic saline (4.5%). CD11b and L-selectin expression on granulocytes from blood and DTT-homogenized sputum were analysed by flow cytometry. Eosinophils could be discriminated from neutrophils by using depolarized light scatter. Disease activity was assessed by baseline FEV1 and airway responsiveness to histamine (PC20). RESULTS: Sputum eosinophils showed higher expression of CD11b (P<0.001) and lower expression of L-selectin (P<0.001) compared with peripheral blood eosinophils. CD11b and L-selectin expression on eosinophils from blood or sputum did not differ between the three groups. Similar results were obtained for neutrophils. The PC20 in the patients with moderate-to-severe asthma was related to CD11b expression on blood (R=-0.92, P=0.001) and sputum eosinophils (R=0.75, P=0.02). CONCLUSIONS: Flow cytometry of induced sputum granulocytes from asthmatic as well as normal subjects is feasible. We conclude that the modulated expression of CD11b and L-selectin on airway granulocytes is not specific for asthmatic airway inflammation, but is probably the result of tissue migration per sé. This implies that CD11b and L-selectin expression on granulocytes in induced sputum cannot be used as marker of disease activity.     

Stimulation of inflammatory markers after blunt trauma

BACKGROUND: Inflammatory mediators are released after trauma and may be related to the pathogenesis of sepsis. A prospective combined study of the pattern of release of an inflammatory mediator, interleukin (IL) 6, leucocyte activation (polymorphonuclear leucocyte (PMN) CD11b receptor expression and plasma elastase-alpha1 proteinase inhibitor complex (E-alpha1PI)) and soluble endothelial adhesion molecule expression (soluble E-selectin (sE-selectin) and soluble intracellular adhesion molecule 1 (sICAM-1)) was performed in patients suffering blunt trauma without adult respiratory distress syndrome (ARDS) or multiple organ failure syndrome (MOFS). METHODS: Thirty-one patients with a mean Injury Severity Score (ISS) of 14 (range 9-57) were studied. Venous blood samples were collected within 6 h of injury and then at 1, 3, 5 and 7 days. Leucocyte CD11b expression was quantified by flow cytometry. Serum IL-6, plasma E-alpha1PI, sE-selectin and sICAM-1 were measured by enzyme-linked immunosorbent assay. RESULTS: Serum IL-6, CD11b expression and E-alpha1PI levels were significantly raised above control values (P < 0.0001) on admission, slowly returning towards control values over the study period (median IL-6, 140 pg/ml versus undetectable; CD11b, 14.8 versus 6.4 mean channel fluorescence units; E-alpha1 PI, 208 versus 52 microg/l). The sICAM-1 level rose to a median of 539 ng/ml at 5 days (control 243 ng/ml). The median sE-selectin level also progressively increased to a maximum level of 80 ng/ml at 5 days (control 49 ng/ml). Eleven patients developed postoperative sepsis. Significant differences in CD11b expression were seen at days 3, 5 and 7 and in E-alpha1 PI at 6 h, 24 h and 3 days in patients who subsequently developed sepsis (P < 0.05). Severe injury (ISS 16 or greater) was associated with significantly greater responses in these measurements. CONCLUSION: These data show that markers of inflammation are specifically stimulated by trauma even when ARDS and MOFS do not occur. The CD11b receptor on PMNs may be useful in screening patients destined to develop post-traumatic sepsis.     

Substance use disorders in a geriatric psychiatry outpatient clinic: prevalence and epidemiologic characteristics

The effects of linoleic acid, linoleic acid anilide, and arachidonic acid on the expression of CD11b/ CD18, CD11c/CD18 integrins and L-selectin on human neutrophils were studied by flow cytometry in a whole blood assay. None of these compounds had any effect on the basal expression of CD11b, CD11c, or L-selectin in the concentration range of 20-100 microM. However, linoleic acid at a concentration of 1000 microM slightly up-regulated CD11b and CD11c by a factor of 2.1 and 1.7, respectively. Linoleic acid, linoleic acid anilide, and arachidonic acid did not affect the formyl-methionyl-leucyl-phenylalanine induced up-regulation of CD11b or CD11c. However, linoleic acid and linoleic acid anilide slightly inhibited the phorbol myristate acetate (PMA)-induced expression of CD11b, which was decreased by 27 and 21% at concentrations of 100 and 1000 microM, respectively. Likewise, arachidonic acid at 40 microM inhibited the PMA-induced expression of CD11b by 19%. Our results suggest that linoleic acid, linoleic acid anilide, and arachidonic acid do not dramatically affect the expression of leukocyte adhesion molecules in a whole blood assay.     

Anti-ICAM-1 monoclonal antibody R6.5 (Enlimomab) promotes activation of neutrophils in whole blood

R6.5 (BIRR-1, Enlimomab), a murine IgG2a mAb to the human ICAM-1, inhibits leukocyte adhesion to the vascular endothelium, thereby decreasing leukocyte extravasation and inflammatory tissue injury. In initial clinical trials, R6.5 proved to be beneficial in reducing both disease activity in refractory rheumatoid arthritis and the incidence of acute rejection after kidney and liver allograft transplantations. However, adverse effects such as fever, leukopenia, or cutaneous reactions were not infrequent. We studied the effects of R6.5 on neutrophil function in whole blood samples ex vivo. Surprisingly, at the concentrations achieved in clinical trials, R6. 5 activated neutrophilic granulocytes, as indicated by a significant increase in expression of the adhesion molecule beta2-integrin CD11b, a concurrent decrease in L-selectin expression, and an enhancement of the oxidative burst activity. Neutrophil activation was not exerted by an anti-ICAM-1 mAb of the IgG1 isotype, by isotype-matched, irrelevant anti-2-phenyloxazolone mAb, or by F(ab')2 fragments of R6.5. Neutrophil activation was completely inhibited by soluble complement receptor type 1. We conclude that in whole blood, R6.5 activates resting neutrophils in a complement-dependent manner. This finding can explain, at least in part, the side effects associated with R6.5 therapy.     

HPMA copolymer bound adriamycin overcomes MDR1 gene encoded resistance in a human ovarian carcinoma cell line

Neutrophils are known to mediate injury in acute ischemic stroke especially during reperfusion. Migration of neutrophils into regions of ischemic injury involves binding to the endothelial cell's intercellular adhesion molecule (ICAM-1) through the leukocyte integrin, CD11/CD18. We studied the potential for neuroprotection with a humanized antibody that binds to and blocks the functions of the CD11/CD18 integrin in a rabbit model of transient focal ischemia. Fifteen New Zealand White rabbits underwent transorbital occlusion of the left middle cerebral, anterior cerebral, and internal carotid arteries using aneurysm clips for 2 h, followed by 6 h of reperfusion. Treatment with a maximally saturating dose (4 mg/kg) of a humanized CD11/CD18 monoclonal antibody (Hu23F2G, ICOS Corp., Bothell, WA) (n = 8) or placebo (n = 7) was administered 20 min after occlusion and given as a single intravenous bolus. Hemispheric ischemic neuronal damage (IND) as seen on hematoxylin- and eosin-stained sections was significantly reduced in Hu23F2G-treated animals by 57% (Hu23F2G: 15 +/- 6.9%; placebo: 35 +/- 5%; mean +/- SEM, P < 0.05, t-test). Immunohistochemical staining with neutrophil elastase confirmed the presence of neutrophils within regions of IND in control brains. Treatment with Hu23F2G resulted in marked reduction of neutrophil infiltration. (No. of neutrophils/IND area: Hu23F2G 36.1 +/- 36.7 cm-2, placebo 460.6 +/- 101.8 cm-2, P = 0.001. ) Antagonism of neutrophil migration at the level of the CD11/CD18 integrin reduces ischemic injury in experimental stroke.     

Functional and phenotypic characterization of monoclonal antibodies to bovine L-selectin

OBJECTIVE: To characterize 2 bovine neutrophil monoclonal antibodies (MAB) as to effects on bovine neutrophil function and their binding antigens on the cell surface of bovine neutrophils. ANIMALS: 16 healthy, lactating Holstein cattle, 1 calf with leukocyte adhesion deficiency, and 1 age-matched control calf, 2 healthy ewes, and 2 healthy human beings as neutrophil sources. PROCEDURE: Neutrophil chemotactic and respiratory burst activities and calcium influx, and binding properties of the 2 MAB were determined. Molecular mass of corresponding cell surface antigens also was determined, as was binding of human L-selectin MAB DREG56 to molecules recognized by MAB 11G10 and 2G8 on the surface of bovine neutrophils. RESULTS: MAB 11G10 and 2G8 inhibited chemotactic activity of bovine neutrophils, up-regulated amplitude of native chemiluminescence, and shortened the time to reach maximal chemiluminescence induced by serum-opsonized zymosan. Crosslinking both MAB with a second antibody induced rapid increase in intracellular free calcium concentration. Binding density of MAB 11G10 and 2G8 to bovine neutrophils treated with trypsin was increased (P < 0.05), compared with that of untreated neutrophils. Neutrophils treated with phosphatidylinositol-specific phospholipase C had decreased (P < 0.05) binding density of MAB 11G10 and 2G8. Binding of the various MAB to neutrophils from calves with bovine leukocyte adhesion deficiency was lower (P < 0.05) than binding to neutrophils from healthy calves. Expression of antigens recognized by the aforementioned MAB on the surface of bovine neutrophils was decreased (P < 0.05) within 10 minutes. CONCLUSION: MAB 11G10 and 2G8 recognized L-selectin molecules on bovine neutrophil membrane. L-Selectin (CD62L) is involved in low-affinity adhesion reactions between leukocytes and L-selectin ligand on postcapillary venular endothelial cells.     

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.     

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.     

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.     

Dialysis neutropenia: the role of the cytoskeleton

Dialysis neutropenia is the result of pulmonary sequestration of neutrophils after complement activation by the dialyzer membrane. Increased expression of neutrophil adhesion receptors, such as CD11b/CD18, suggests that neutrophil adhesion to the capillary endothelium is a possible mechanism. An alternative hypothesis is that the complement fragment C5a modulates neutrophil mechanical properties via the cytoskeleton-largely filamentous actin (F-actin)-stiffening them and thereby slowing their passage through the pulmonary capillaries. To investigate this hypothesis, we developed an assay to measure the F-actin content of neutrophils in whole blood using flow cytometry and the stain NBD-phallacidin. We measured neutrophil F-actin content during hemodialysis of patients with polysulfone (N = 6), Hemophan (N = 6), and Cuprophan membranes sterilized with either ethylene oxide (N = 5) or steam (N = 6). Cell counts, neutrophil and monocyte CD11b expression and plasma C5a concentrations were also measured. The results confirm the strong relationship between the degree of neutropenia, increases in CD11b expression and plasma C5a levels reported by previous researchers. Modulation of the F-actin content of neutrophils was also strongly related to C5a levels, indicating that the neutrophil cytoskeleton is active during dialysis. Modeling of cell counts suggests that with Cuprophan a substantial fraction of neutrophils and monocytes are sequestered before they even pass through the dialyzer, suggesting some form of systemic activation of these cells. Evidence for systemic activation was also seen in measurements of F-actin content, but not CD11b expression, a finding that strengthens the case for the involvement of the cytoskeleton in dialysis neutropenia.     

Adhesion of leukocytes to endothelial cells in atherosclerosis]

The adherence of blood monocytes to the endothelium, followed by transmigration beneath the endothelium, are initiating events in the formation of foam cells, promoting atherogenesis. We showed that adhesion molecules on leukocytes were up- or down-regulated in atherosclerosis, when binding of monoclonal antibodies was measured by indirect immunofluorescence with flow cytometry. Expression of PE-CAM-1 (CD31) on monocytes and LFA-1 (CD11a) on lymphocytes was increased with age. Expression of PECAM-1 in monocytes was also up-regulated in patients with coronary artery disease. Being unchanged on aging, expression of HAR (CD44) on polymorphonuclear leukocytes and monocytes was increased in patients with coronary artery disease. On the other hand, expression of L-selectin (CD62L) on polymorphonuclear leukocytes, and LFA-1, CR3 (CD11b) and VLA-4 (CD49d) on monocytes was decreased. These findings may show the mechanism of increased chemotaxis of monocytes beneath the endothelium during the incipient stage of atherosclerosis.     

Obstructive jaundice causes reduced expression of polymorphonuclear leucocyte adhesion molecules and a depressed response to bacterial wall products in vitro

BACKGROUND: Obstructive jaundice is associated with an increased incidence of infection and endotoxaemia, which may result from impaired host immunity. Neutrophil adhesion to vascular endothelium is a key part of the inflammatory response. AIMS: To investigate neutrophil adhesion molecule expression and activation in obstructive jaundice. PATIENTS: Nine adult patients with obstructive jaundice and 11 control subjects. METHODS: The expression of the neutrophil adhesion receptors L-selectin, CD11a, CD11b, CD11c, and CD15 was determined using flow cytometry. CD11b expression in response to stimulation with fMLP and endotoxin was measured. RESULTS: The basal expression of L-selectin, CD11a, and CD15 was significantly decreased in jaundiced patients (p < 0.05) and the expression of CD11b in response to stimulation with fMLP and endotoxin was significantly impaired in the jaundiced group. Endotoxin stimulation without plasma did not reverse the impaired response showing that it is not caused by endotoxin inactivation by plasma proteins. CONCLUSIONS: Neutrophils from patients with obstructive jaundice show decreased adhesion receptor expression and an impaired response to stimulation with bacterial products. This cellular dysfunction may be responsible for the high incidence of septic complications in these patients.     

Cytokine-regulated expression of activated leukocyte cell adhesion molecule (CD166) on monocyte-lineage cells and in rheumatoid arthritis synovium

OBJECTIVE: To determine whether monocyte/macrophage expression of the CD6 ligand, activated leukocyte cell adhesion molecule (ALCAM) (CD166), is regulated by cytokines during inflammation in rheumatoid arthritis (RA). METHODS: We used flow cytometry to test whether cytokines present in rheumatoid synovium could regulate ALCAM cell surface expression on peripheral blood (PB) monocytes and RA synovial fluid (SF) macrophages, and we examined ALCAM expression in situ in RA synovium by immunofluorescence. RESULTS: The monocyte differentiation factors interleukin-3, macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor augmented ALCAM expression on PB monocytes. ALCAM was expressed on monocyte-lineage cells in situ in inflamed synovium from patients with RA (9 of 9), but not in uninflamed synovium from patients with joint trauma (0 of 3). Furthermore, in vitro culture-induced ALCAM expression on PB monocytes and CD14+ RA SF cells was inhibited by an M-CSF neutralizing antibody. CONCLUSION: ALCAM expression on PB and SF monocytes/macrophages is enhanced by M-CSF.