Increased Expression of Leucocyte Adherence-Related Glycoproteins by Polymorphonuclear Leucocytes during Phagocytosis of Staphylococci on an Endothelial Surface

Phagocytosis of Staphylococcus aureus by human polymorphonuclear leucocytes (PMN) on the surface of endothelial cells is accompanied by adherence of the PMN to the endothelial surface and detachment of the endothelial cells from the culture monolayer. We studied the role of the leucocyte adherence-related glycoproteins (Leu-CAM: Mo1/LFA-1/150,95 or CD11a-c-CD18 complex) in these processes. Phagocytosis of S. aureus induced increased expression of the common beta chain (CD18) of Leu-CAM as demonstrated by flow cytometric analysis of PMN treated with a monoclonal antibody (MoAb) (CLB-LFA-1/1) directed against CD18 and fluorescein isothiocyanate (FITC)-conjugated anti-MoAb. This same MoAb also inhibited the increased adherence of the PMN to the endothelial cells which occurs during phagocytosis. Blocking of adherence during phagocytosis with MoAb CLT-LFA-1/1 had no effect on the detaching activity of the PMN on the endothelial cells. We conclude that adherence of PMN to endothelial cells during phagocytosis of S. aureus is mediated by the Leu-CAM complex. Adherence through the Leu-CAM, however, is not necessary for endothelial damage by the phagocytosing PMN.     

Altered expression of CD11/CD18 on the peripheral blood phagocytes of patients with tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is characterized by granulomatous lesions made up of epithelioid cells, giant cells and mononuclear leucocytes. Cell-cell adhesion is important in granuloma formation and in the leucocyte migration which accompanies it. We have recently shown increased expression of the adhesion molecules CD11/CD18 (LeuCAMs, beta 2 integrins) on peripheral blood leucocytes from patients with sarcoidosis (Shakoor & Hamblin, 1992). Here we have studied the expression of CD11/CD18 and CD29 (VLA beta 1 integrin) on the peripheral blood leucocytes of 10 TB patients by flow cytometry. The density (expressed as mean fluorescence intensity) of CD11b on monocytes and polymorphs was increased (P < 0.005), as was CD11c (P < 0.005) and CD18 (P < 0.05) on polymorphs. CD11a expression was significantly reduced on polymorphs (P < 0.05). No differences were found in the expression of CD29, the percentages of cells expressing any molecule and, in contrast to sarcoidosis, the density of any molecule on lymphocytes. Although the cytokine tumour necrosis factor (TNF) has been implicated in the process of up-regulation, an ELISA for TNF failed to detect significant levels in plasma. The results suggest increased peripheral phagocyte CD11/CD18 expression is a feature of TB, which may contribute to the pathological processes involved.     

Cerebral leucocyte infiltration in lupus-prone MRL/MpJ-fas lpr mice--roles of intercellular adhesion molecule-1 and P-selectin

The autoimmune disease which affects MRL/MpJ-fas(lpr) mice results in cerebral leucocyte recruitment and cognitive dysfunction. We have previously observed increased leucocyte trafficking in the cerebral microcirculation of these mice; however, the types of leucocytes recruited have not been analysed thoroughly, and the roles of key endothelial adhesion molecules in recruitment of these leucocytes have not been investigated. Therefore the aim of this study was to classify the phenotypes of leucocytes present in inflamed brains of MRL/MpJ-fas(lpr) mice, and dissect the roles of endothelial adhesion molecules in their accumulation in the brain. Immunohistochemical analysis revealed significant leucocyte infiltration in the brains of 16- and 20-week-old MRL/MpJ-fas(lpr) mice, affecting predominantly the choroid plexus. Isolation of brain-infiltrating leucocytes revealed that lymphocytes and neutrophils were the main populations present. The CD3(+) lymphocytes in the brain consisted of similar proportions of CD4(+), CD8(+) and CD4(-)/CD8(-)[double negative (DN)] populations. Assessment of MRL/MpJ-fas(lpr) mice deficient in endothelial adhesion molecules intercellular adhesion molecule-1 (ICAM-1) or P-selectin indicated that cerebral leucocyte recruitment persisted in the absence of these molecules, with only minor changes in the phenotypes of infiltrating cells. Together these data indicate that the brains of MRL/MpJ-fas(lpr) mice are affected by a mixed leucocyte infiltrate, of which the unusual DN lymphocyte phenotype contributes a substantial proportion. In addition, endothelial adhesion molecules ICAM-1 and P-selectin, which modulate survival of MRL/MpJ-fas(lpr) mice, do not markedly inhibit leucocyte entry into the central nervous system.     

Decreased expression levels of L-selectin on subsets of leucocytes and increased serum L-selectin in severe psoriasis

L-selectin is a leucocyte adhesion molecule involved in leucocyte interactions with vascular endothelial cells. Following leucocyte activation L-selectin is endoproteolytically released from the cell surface. To assess whether psoriasis vulgaris results in systemic leucocyte activation, we examined expression levels of L-selectin on subsets of peripheral blood leucocytes from patients with psoriasis (n = 25) and normal control subjects. Serum levels of soluble L-selectin were quantified by ELISA in patients with psoriasis (n = 75), pustulosis palmaris et plantaris, and contact dermatitis, as well as normal control subjects. Psoriasis severity was evaluated by psoriasis area and severity index (PASI). L-selectin expression levels on CD4+ T cells, B cells, monocytes, and neutrophils from patients with severe-type psoriasis (PASI > or = 15) was significantly decreased compared with leucocytes from normal control subjects. Furthermore, L-selectin expression on CD4+ T cells showed good inverse correlation with PASI scores. Monocyte L-selectin expression was restored when the skin lesions of psoriasis were remitted. The frequencies of L-selectin+ CD4+ T cells or L-selectin+ CD8+ T cells from patients with psoriasis were almost normal. Serum L-selectin levels in patients with severe-type psoriasis were significantly higher than those in normal control subjects. These results suggest that subsets of leucocytes may be activated in psoriasis, and that L-selectin expression levels on some leucocyte subsets, especially CD4+ T cells, tend to correlate with disease severity of psoriasis.     

Blood Leucocyte Subsets of the Rat: Expression of Adhesion Molecules and Localization Within High Endothelial Venules

Although several distinct adhesion pathways are now well characterized, it is not clear whether analysis of adhesion molecule expression on leucocytes is sufficient to predict their interaction with endothelium in vivo. Therefore, in the present study this question was addressed by investigating the interaction between blood leucocyte subsets and high endothelial venules (HEV). The expression of different types of adhesion molecule (CD44, α4-integrins, LFA-1, ICAM-1, CD2 and L-selectin) on lymphocytes, NK cells, monocytes and granulocytes of rat blood was determined by flow cytometry. In the same animals the numbers of blood leucocyte subsets present in the HEV of axillary lymph nodes and Peyer's patches were analysed using immunohistology. In the HEV of both axillary lymph nodes and of Peyer's patches lymphocytes (> 10.000 per mm²), as well as small numbers of NK cells and monocytes (< 500 per mm²), were found. In contrast, granulocytes were not detected here. Lymphocytes, NK cells, monocytes and granulocytes each expressed CD44, α4-integrins, LFA-1, ICAM-1, CD2 and L-selectin in a pattern characteristic to cell type, but this did not correlate with the different ability of the leucocyte subsets to interact with the two types of HEV. In conclusion, determining the expression of CD44, α4-integrins, LFA-1, ICAM-1, CD2 and L-selectin on blood leucocytes alone is not sufficient to predict leucocyte/endothelium interaction in vivo     

Detection of small numbers of immature cells in the blood of healthy subjects

AIMS: To determine the frequency of immature haemopoietic cells in the peripheral blood of healthy persons. METHODS: Cytocentrifuge preparations were made using mononuclear leucocytes separated by a Ficoll-Hypaque density gradient. The slides were stained by May-Grünwald-Giemsa. The combination with immunoperoxidase technique allowed immunotyping of uncommon blood cells. RESULTS: Blast cells expressing the progenitor cell marker CD34 represented 0.11 (0.06) per cent (mean (SD)) of the total mononuclear leucocyte count; these were the haemopoietic progenitor cells in the peripheral blood. Dark blue cells expressing CD38, CD45, HLA-DR, CD4, CD11a, CD29, CD49d, CD50, and CD54 represented 0.30 (0.21) per cent of the mononuclear leucocytes; most of these cells did not express T, B, NK, myelomonocytic, progenitor cell, proliferation, activation, blood dendritic cell, or follicular dendritic cell markers. These were dendritic cell precursors in the peripheral blood. Very small numbers of cells expressing CD83 were found. Blast-like cells expressing CD45, HLA-DR, CD11a, and CD50 represented 0.15 (0.10) per cent of the mononuclear leucocytes; morphology and immunotyping supported the conclusion that these cells were poorly differentiated monocytes. CONCLUSIONS: Morphological investigation of mononuclear leucocytes in peripheral blood of healthy persons can be used to detect small numbers of blasts, dark blue cells, and blast-like cells. The immunoperoxidase technique can then be used for immunotyping of these cells. This simple method may be helpful in diagnosing haematological disorders.     

Porins and lipopolysaccharide (LPS) from Salmonella typhimurium induce leucocyte transmigration through human endothelial cells in vitro.

Bacteria or bacterial products may constitute important inducers of surface molecule expression on endothelial cells and leucocytes. This study was undertaken to determine the effects of the Salmonella typhimurium porins, LPS-S and LPS-R on the transendothelial migration of leucocytes through human umbilical vein endothelial cells (HUVEC). Treatment of the HUVEC with either porins or LPS-S or LPS-R increased the transmigration of different leucocyte populations, in particular that of neutrophils. The maximal increase occurred using LPS-S treatment, whereas porin stimulation fell between LPS-S and LPS-R. The transmigration increase was dose-dependent and reached its maximum at about 100-1000 ng/ml of stimulus. Optimal endothelial activation occurred after 2-4 h and 4-6 h using LPS and porin, respectively. Stimulation of leucocytes with either porins or LPS slightly increased their transmigration through non-activated endothelial cells. Transmigration increased remarkably during the simultaneous stimulation of endothelial cells by IL-1ss together with either porins or LPS. To assess participation of E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and leucocyte adhesion complex (CD11/18) in porin- or LPS-mediated leucocyte migration, blocking MoAbs were used. Each blocking MoAb partially and selectively decreased leucocyte transmigration. The obtained results contribute to clarify some aspects of the inflammatory process at sites of infection.     

Role of leucocytes in damage to the vascular endothelium during ischaemia-reperfusion injury

During this investigation, a model of tourniquet-induced forearm ischaemia-reperfusion injury is employed to investigate the role of leucocytes in damage to the vascular endothelium during ischaemia-reperfusion injury. Leucocyte entrapment is investigated by measuring the concentration of leucocytes in venous blood leaving the arm. Neutrophil and monocyte leucocyte subpopulations are isolated by density gradient centrifugation techniques. Cell surface expression of CD11b and the intracellular production of hydrogen peroxide are measured via flow cytometry. Plasma concentrations of elastase and von Willebrand factor (vWF) are measured using enzyme-linked immunosorbemt assay (ELISA) techniques. During ischaemia-reperfusion, there was an increase in CD11b cell surface expression on neutrophils (P=0.040) and monocytes (P=0.049), and a decrease in peripheral blood leucocytes (P=0.019). There was an increase in the intracellular production of hydrogen peroxide by leucocyte subpopulations (P=0.027 [neutrophils], P=0.091 [monocytes]) and in the plasma elastase concentration (P=0.05). There was also a trend to increasing plasma concentration of vWF (P=0.0562), which was measured as a marker of endothelial damage. Ischaemia-reperfusion results in increased adhesiveness, entrapment and activation of leucocytes. Even following a mild ischaemic insult, this leucocyte response was followed immediately by evidence of endothelial damage. These results may have important implications for understanding the development of chronic diseases that involve mild ischaemic episodes.     

Increased CD11/CD18 expression on the peripheral blood leucocytes of patients with HIV disease: relationship to disease severity.

In HIV disease increased adhesion between leucocytes themselves and between leucocytes and endothelium may contribute to cell loss and viral spread. Using a novel method for the preparation of blood leucocytes for flow cytometry, we report increased expression of leucocyte adhesion molecules (LeuCAMs) (CD11/CD18) on peripheral blood leucocytes of patients with HIV disease compared with normal controls. Patients were divided into two groups on the basis of CD4 T lymphocyte numbers (those with > 0.5 x 10(9)/l and those with < 0.2 x 10(9)/l), and assessed for p24 antigen expression, viral load and serum tumour necrosis factor (TNF) levels as well as LeuCAM expression. Patients with < 0.2 x 10(9)/lCD4 cells had more p24 antigen and more HIV infectious virus and more serum TNF than those with > 0.5 x 10(9)/l. Whilst the percentages of only monocytes and polymorphs expressing CD11b were significantly increased in patients with the least CD4 cells, the density of LeuCAMs, expressed as mean fluorescence intensity (MFI), was significantly increased on all leucocytes, with the most significant increases being seen on patients with the fewest CD4 T cells. Our findings are consistent with leucocyte activation by a soluble factor, although we could find no correlation between levels of TNF and LeuCAM expression. The increased expression of adhesion molecules on peripheral blood leucocytes could play a role in the cellular extravasation and aggregation seen in HIV disease.     

Impaired leucocyte rolling,adhesion and transendothelial migration following cuprophane haemodialysis

THORLACIUS, H., FERNVIK, E., GAUTAM, N., LUNDAHL, J., RAUD, J., JACOBSON, S.H. & LINDBOM, L. 1997. Impaired leucocyte rolling, adhesion and transendothelial migration following cuprophane haemodialysis. Acta Physiol Scand 159 , 277–283, Received 29 July 1996, accepted 13 November 1996. ISSN 0001–6772. Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden. In this study, we investigated how different steps in the extravasation process of leucocytes, i.e. rolling, adhesion and transendothelial migration, are affected by haemodialysis with cuprophane membranes. Human leucocytes obtained from whole blood prior to clinical haemodialysis and from the afferent blood line (post-dialyser) 15 min after the initiation of dialysis were injected into the mesenteric microcirculation of urethane anaesthetized rabbits and analysed for their ability to roll in the microvessels by use of intravital fluorescence microscopy. Moreover, neutrophils from the two leucocyte populations were compared with respect to chemoattractant-induced adhesion and transmigration across confluent monolayers of bovine aortic endothelial cells. Our results show that, as compared with pre-dialysis leucocytes, 15 min of cuprophane haemodialysis impaired leucocyte rolling by 78 ± 7%, reduced N-formyl-methionyl-leucyl-phenylalanin (fMLP)-induced adhesion by 34 ± 9%, and abolished transendothelial migration. These findings demonstrate that intradialytical activation of leucocytes during cuprophane haemodialysis severely affects leucocyte functions that are critical in the extravasation process of these cells at inflammatory tissue sites, and thus may help explain the increased susceptibility to infections observed in patients on chronic haemodialysis.     

Identical expression of ELAM-1, VCAM-1, and ICAM-1 in sarcoidosis and usual interstitial pneumonitis

Extravasation of leucocytes in tissues is mediated by leucocyte—endothelial cell interactions in which adhesion molecules play an important role. Until now, two pathways have been unravelled, i.e., the LFA-1/ICAM-1 and the VLA-4/VCAM-1 pathways. ELAM-1 has been shown to be involved in granulocyte accumulation and recently also in lymphocyte migration. The role of HECA-452 is under investigation. In this study we have investigated the expression of the above-mentioned adhesion molecules in lung tissue of patients with pulmonary sarcoidosis and usual interstitial pneumonitis (UIP), and in mediastinal lymph nodes of patients with sarcoidosis. ICAM-1 (CD54) was broadly distributed on the endothelium of all the vessels found in sarcoidosis and UIP. VCAM-1 was present on the endothelium of the venules, capillaries, and arterioles in both sarcoidosis and UIP. ELAM-1 reacted with endothelial cells lining venules and capillaries in chronic progressive sarcoidosis and in the active phase of UIP but not in the stationary phases of both diseases. HECA-452 activity could be detected only on high endothelial venules within sarcoid lymph nodes. In lung tissues, macrophages bearing the ICAM-1 antigen were present in sarcoid tissue but not in the interstitium and alveolar space of UIP. LFA-1 (CD11a/CD18) and VLA-4 (CD49d/CD29) were present on all leucocytes found but seemed to be more highly expressed on lymphocytes in sarcoidosis. These findings suggest that the LFA-1/ICAM-1 and VLA-4/VCAM-1 pathways are involved in leucocyte migration in both types of lung disease, while in the active phases of sarcoidosis and UIP, ELAM-1 is also involved.     

Enhanced expression of L-selectin on peripheral blood lymphocytes from patients with IgA nephropathy

To investigate the homing characteristics of T and B lymphocytes which could explain the abnormal partition of IgA-producing cells in tonsils and bone marrow from patients with IgA nephropathy (IgAN), the expression of leucocyte adhesion molecules (CD11a, CD29, CD49d, CD62L, CD31) was assessed using flow cytometry on peripheral blood leucocytes from patients with biopsy-proven IgAN and controls. Higher proportions of T and B lymphocytes expressing higher amounts of L-selectin, as well as higher proportions of B cells expressing more CD31 were evidenced in IgAN patients. Conversely, serum levels of sCD62L were not different from controls, but significantly higher than serum levels in patients suffering from other renal diseases. We hypothesize that this over-expression of CD62L and CD31 may be involved in an enhanced efficiency of lymphoid cells homing to lymphoid tissues in this disease.     

Increased CD11/CD18 expression on peripheral blood leucocytes of patients with sarcoidosis.

Sarcoidosis is a multisystem disease of unknown etiology characterized by non-caseating granulomata, formed mainly from macrophages surrounded by lymphocytes and plasma cells. Using a novel method for the preparation of blood leucocytes for flow cytometry, we report increased expression of LeuCAMs (CD11/CD18) on peripheral blood leucocytes of 11 Caucasian and 10 Afro-Caribbean patients with sarcoidosis compared with age-, sex- and race-matched controls. Whilst the percentages of the cells expressing CD11/CD18 were no different, the density, expressed as mean fluorescence intensity (MFI), was greater for all leucocytes in sarcoids than in normal individuals. The expression of intercellular adhesion molecule-1 (ICAM-1), a ligand for LFA-1 which is expressed on all leucocytes, was not significantly different from normal, whereas HLA-DR was expressed more intensely on sarcoid monocytes (P less than 0.01) and blood lymphocytes (P less than 0.005) than control cells. Our findings are consistent with leucocyte activation although we were unable to confirm reports of elevated tumour necrosis factor-alpha (TNF-alpha) in the patients' plasma using an ELISA. Increased expression of adhesion molecules on peripheral blood leucocytes may play a role in the cellular extravasation, aggregation, and granuloma formation seen in sarcoidosis.     

Regulation of Intercellular Adhesion Molecule-1 Expression on Endothelial Cells with Correlation to Lymphocyte-Endothelial Binding

Intercellular adhesion molecule-1 (ICAM-1) expression on cultured human umbilical cord vein endothelial cells (EC) and its correlation to the leucocyte adherence to EC was investigated. Gamma interferon (IFN-gamma) increases the ICAM-1 expression on EC and concomitantly also increases the binding of leucocytes to EC in vitro. Methylprednisolone down-regulates the IFN-gamma-induced binding, but does not alter the ICAM-1 expression. Leukotriene B4 (LTB4) rapidly increases the binding of lymphocytes to EC, but does not induce ICAM-1 expression on the EC. The dissociation between ICAM-1 expression and leucocyte binding to EC clearly indicates that ICAM-1 is not the only ligand.     

An abnormal adherence of monocytes to fibronectin in thyroid autoimmunity has consequences for cell polarization and the development of veiled cells

Blood monocytes of patients with thyroid autoimmune disease (TAID) display defects in rearranging their cortical actomyosin cytoskeleton ('polarize') in response to chemoattractants. Such rearrangements also take place after the adherence of monocytes to the extracellular matrix (ECM). It is therefore not surprising that monocytes are primed after fibronectin (FN) adherence, displaying an enhanced polarization toward chemoattractants. We investigated the integrin expression and chemoattractant-induced polarization of monocytes of TAID patients before and after FN adherence. Since cytoskeletal rearrangements are also required during the transition of monocytes into veiled antigen-presenting cells (VCs), we investigated such transition of FN-adherent monocytes of TAID patients. Adherent and nonadherent monocyte populations from TAID patients and healthy controls were subjected to a polarization test with the chemoattractant fMLP (or MCP-1), FACS analyses (FITC-labelled FN, CD29, CD49e, d, b and a) and tested for their capability to develop into veiled APC. Monocytes of healthy individuals showed an improved chemoattractant-induced cell polarization after FN adherence, not reflected by TAID monocytes, in which chemoattractant-induced polarization worsened. Monocytes of healthy individuals up-regulated CD49e and d integrins and their capability to bind FITC-labelled FN after adherence to a FN-coated plate, as well as enhancing their capability to generate T cell-stimulatory VCs. Monocytes of TAID patients did not. These data indicate that integrin- (and chemokine-) mediated functions are hampered in monocytes of TAID patients. Because integrin action is pivotal to processes such as monocyte adherence to endothelial cells, uropod formation, migration into tissues and differentiation into APC and macrophages, these defects might underly immune dysbalances important in thyroid autoimmune development.     

Molecules mediating adhesion of T and B cells, monocytes and granulocytes to vascular endothelial cells.

Leucocytes interact with vascular endothelial cells (EC), and adhesion between these two cell types in vitro is modulated by phorbol ester. Monocytes were found to display the highest basal adhesion to EC, followed by Epstein-Barr virus-immortalized normal B cells (EBV-B), T cells and granulocytes. Phorbol ester treatment increased the adhesion of all types of leucocytes, except monocytes. In the presence of this compound, monoclonal antibody 60.3 to GP90 (CD18, a leucocyte-adhesion protein which is non-covalently associated to either GP160, GP155, or GP130) was found to inhibit the adhesion of the four types of leucocytes to a considerable extent, while anti-lymphocyte function-associated antigen-1 (LFA-1) antibody to GP160 (CD11a) inhibited the adhesion of T and B cells only. Antibody 60.1 to GP155 (CD11b) had a major inhibitory activity exclusively on granulocytes, while antibody LB-2, which recognizes a distinct adhesion molecule (GP84) and, in contrast to the previous antibodies, reacts with EC, mainly inhibited adhesion of EBV-B and did not increase the inhibition obtained with antibody 60.3 alone. Fab fragments of antibody 60.3 inhibited leucocyte adhesion more efficiently, in either the absence or presence of phorbol ester, than the intact antibody molecule. It is concluded the GP90, either alone or associated to the larger glycoproteins, mediates the adhesion in all types of leucocytes, while GP84 mediates the adhesion of the activated B cells.     

The Marginal Blood Pool of the Rat Contains not only Granulocytes, but also Lymphocytes, NK-Cells and Monocytes: a Second Intravascular Compartment, its Cellular Composition, Adhesion Molecule Expression and Interaction with the Peripheral Blood Pool

To leave the blood, leucocytes marginate to the vessel wall. Granulocytes thereby form the so-called marginal pool. It is unclear to what extent such a second intravascular compartment also exists for lymphocyte subsets, NK-cells and monocytes. Samples of the peripheral blood and the marginal pool of the LEW rat were analysed by flow cytometry. In the marginal pool the percentage of granulocytes and monocytes was significantly higher compared to that of the peripheral blood, and the proportion of 'naive' T and B lymphocytes was decreased. The expression of LFA-1 was higher on all leucocyte subsets of the marginal pool except the granulocytes, whereas no differences were seen for the expression of other adhesion molecules (α₄-integrins, ICAM-1, CD2, L-selectin, and CD44). In addition, splenectomy influenced the cellular composition of peripheral blood and marginal pool differently and, after injection of blood leucocytes, these cells were found in both compartments showing its characteristic cellular composition. Thus, not only granulocytes, but also B and T lymphocyte subsets, NK-cells and monocytes form a second distinct intravascular compartment. This marginal pool probably influences the cellular composition of leucocyte subsets available for entry into the tissues.     

Platelet endothelial cell adhesion molecule-1 is expressed in adenoidal crypt epithelial cells

The adenoidal epithelial crypt is a potential site of antigen transport from pharyngeal lumen to adenoidal tissue. The base of the crypt is consistently infiltrated with leucocytes, forming a reticular lymphoepithelial structure. To evaluate mechanisms that possibly mediate leucocyte infiltration, expressions of leucocyte adhesion molecules, such as platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31), vascular cell adhesion molecule-1 (VCAM-1) (CD106) and intercellular adhesion molecule-1 (ICAM-1) (CD54), were studied in the adenoidal epithelial crypt. Epithelial cells in the outer opening of the adenoidal crypt were positive for VCAM-1, whereas epithelial cells at the base of the crypt were positive for PECAM-1. Isolated ICAM-1-expressing cells were found throughout the epithelial crypt. Double immunofluorescence staining revealed that the epithelial cells positive for PECAM-1 or VCAM-1 were positive for cytokeratin. The expression of PECAM-1 in the base and VCAM-1 at the orifice of the adenoidal epithelial crypt implies that the base and the orifice of the crypt have a distinct ability to recruit leucocytes. Epithelial cells expressing PECAM-1 may have a role in the formation of the reticular lymphoepithelial structure in the epithelial crypt.     

Leucocyte-induced endothelium-dependent vasodilatation and post-ischaemic vasospasm in the isolated rat superior mesenteric artery

The purpose of this study was to investigate the possible role of leucocytes in the pathogenesis of reperfusion-induced vasospasm of ischaemic mesenteric arteries. Scanning electron microscopy of the rat superior mesenteric artery (SMA) after 30 min of ischaemia in vivo revealed adherence of leucocytes to the vessel wall. Isolated SMA preparations were perfused with Krebs-Henseleit buffer containing noradrenaline. Infusion of homologous leucocytes resuspended in perfusate (3 x 10(6) cells/ml) into these preconstricted preparations caused a fall in resistance of 29 +/- 2%. Removal of the endothelium by collagenase treatment abolished this response. Indeed, leucocyte infusion caused an increase in resistance of 39 +/- 8% under these circumstances. Following 30 min of normothermic ischaemia, leucocyte infusion caused a transient vasodilatation of 31 +/- 4% followed by an increase of 38 +/- 11% in the perfusion resistance of isolated SMA preparations. In each case, a similar response was obtained to infusion of the cell-free supernatant. These results suggest that leucocyte activation occurs in vivo during reperfusion of the SMA after as little as 30 min of ischaemia, and that activated leucocytes can release humoral vasoactive factors which evoke an endothelium-dependent vasodilator response in normal vessels but a predominantly vasoconstrictor response following brief intervals of ischaemia.     

Adhesion molecules are upregulated on dendritic cells isolated from human blood.

This study investigated whether the high expression of adhesion molecules on enriched preparations of circulating dendritic cells (DCs) was an intrinsic property of the cells or whether it was a consequence of the procedure used to isolate them from blood. Expression of the beta 1, beta 2 integrins (CD11/CD18 family) and other adhesion molecules on DCs in whole blood was compared with that on isolated DCs. Dendritic cells were identified by flow cytometry as leucocytes that were positive for human leucocyte antigen (HLA)-DR, but negative for CD3, CD14, CD16, CD19 and CD56. In contrast to a minority of DCs in whole blood, the majority of isolated DCs expressed the beta 2 integrins and there were a greater number of cells bearing CD44, CD54 and some of the beta 1 integrins (notably CD49b, CD49d, CD49e and CD29). An increase in the proportion of DCs bearing adhesion molecules was generally apparent at the isolation stage when mononuclear cells, which had been incubated overnight, were centrifuged on a metrizamide gradient to enrich for cells of low density. Inclusion of an inhibitor of protein glycosylation and exocytosis (brefeldin A) at all stages of separation partially prevented an increase in the percentage of DCs bearing CD18, C29 and C54 whereas the inclusion of cycloheximide (an inhibitor of polypeptide synthesis) interfered with increases in the percentage of cells bearing CD29 and CD54. Neither of these antagonists had an effect on the intensity of adhesion molecule expression. We suggest that some of the adhesion-dependent functions of isolated DCs are caused, in part, by an upregulation of surface adhesion molecules induced by the enrichment procedure.