α3ß1 integrins regulate CD151 complex assembly and membrane dynamics in carcinoma cells within 3D environments.

Integrins are extracellular matrix (ECM) receptors that are key players in the regulation of tumour cell invasion. The laminin-binding integrin α3ß1 has previously been shown to regulate adhesion and migration of carcinoma cells in part through co-operative signalling with the tetraspanin family of transmembrane proteins. However, the spatial and temporal regulation of crosstalk between these families of transmembrane proteins in intact cells remains poorly understood. Here we have used fluorescence resonance energy transfer (FRET) to demonstrate for the first time that α3ß1 and the tetraspanin CD151 directly associate at the front and retracting rear of polarised migrating breast carcinoma cells in both two-dimentional (2D) and three-dimentional (3D)matrices. Furthermore, localised α3ß1-CD151 binding correlates with lower CD151 homodimerisation in cells migrating on laminin or within matrigel. Loss of α3ß1 integrin leads to increased CD151 homodimer formation, increased activation of Rho GTPase, loss of cell polarity and decreased invasion in 3D ECM. As a result, α3-silenced cells show decreased actin-based membrane protrusion and retraction in both 2D and 3D environments. These data demonstrate that associations between α3ß1 and CD151 occur dynamically within discrete subcellular compartments and act to establish local GTPase signalling to promote tumour cell invasion. These novel findings shed light on the complex crosstalk and switching between receptor complexes in response to different extracellular cues during cell invasion in 3D environments.

A role for iron in Wnt signalling.

There is an emerging body of evidence implicating iron in carcinogenesis and in particular colorectal cancer, but whether this involves Wnt signalling, a major oncogenic signalling pathway has not been studied. We aimed to determine the effect of iron loading on Wnt signalling using mutant APC (Caco-2 and SW480) and wild-type APC (HEK-293 and human primary fibroblasts) containing cell lines. Elevating cellular iron levels in Caco-2 and SW480 cells caused increased Wnt signalling as indicated by increased TOPFLASH reporter activity, increased mRNA expression of two known targets, c-myc and Nkd1, and increased cellular proliferation. In contrast wild-type APC and beta-catenin-containing lines, HEK 293 and human primary fibroblasts were not responsive to iron loading. This was verified in SW480 cells that no longer induced iron-mediated Wnt signalling when transfected with wild-type APC. The cell line LS174T, wild type for APC but mutant for beta-catenin, was also responsive suggesting that the role of iron is to regulate beta-catenin. Furthermore, we show that E-cadherin status has no influence on iron-mediated Wnt signalling. We thus speculate that excess iron could exacerbate tumorigenesis in the background of APC loss, a common finding in cancers.

Modulation of iron transport proteins in human colorectal carcinogenesis.

BACKGROUND AND AIMS: Total body iron and high dietary iron intake are risk factors for colorectal cancer. To date there is no comprehensive characterisation of iron transport proteins in progression to colorectal carcinoma. In this study, we examined expression of iron import (duodenal cytochrome b (DCYTB), divalent metal transporter 1 (DMT1), and transferrin receptor 1 (TfR1)) and export (hephaestin (HEPH) and ferroportin (FPN)) proteins in colorectal carcinoma. METHODS: Perl's staining was used to examine colonocyte iron content. Real time polymerase chain reaction (PCR) and western blotting were used to examine mRNA and protein levels of the molecules of interest in 11 human colorectal cancers. Semiquantitative immunohistochemistry was used to verify protein levels and information on cellular localisation. The effect of iron loading on E-cadherin expression in SW480 and Caco-2 cell lines was examined by promoter assays, real time PCR and western blotting. RESULTS: Perl's staining showed increased iron in colorectal cancers, and there was a corresponding overexpression of components of the intracellular iron import machinery (DCYTB, DMT1, and TfR1). The iron exporter FPN was also overexpressed, but its intracellular location, combined with reduced HEPH levels, suggests reduced iron efflux in the majority of colorectal cancers examined. Loss of HEPH and FPN expression was associated with more advanced disease. Iron loading Caco-2 and SW480 cells caused cellular proliferation and E-cadherin repression. CONCLUSIONS: Progression to colorectal cancer is associated with increased expression in iron import proteins and a block in iron export due to decreased expression and aberrant localisation of HEPH and FPN, respectively. This results in increased intracellular iron which may induce proliferation and repress cell adhesion.

Glucocorticoid augmentation of macrophage capacity for phagocytosis of apoptotic cells is associated with reduced p130Cas expression, loss of paxillin/pyk2 phosphorylation, and high levels of active Rac.

Phagocytic clearance of apoptotic granulocytes has a pivotal role in determining an inflammatory outcome, resolution or progression to a chronic state associated with development of fibrotic repair mechanisms, and/or autoimmune responses. In this study, we describe reprogramming of monocyte to macrophage differentiation by glucocorticoids, resulting in a marked augmentation of their capacity for phagocytosis of apoptotic neutrophils. This monocyte/macrophage phenotype was characterized by decreased phosphorylation, and therefore recruitment of paxillin and pyk2 to focal contacts and a down-regulation of p130Cas, a key adaptor molecule in integrin adhesion signaling. Glucocorticoid-treated cells also displayed higher levels of active Rac and cytoskeletal activity, which were mirrored by increases in phagocytic capability for apoptotic neutrophils. We propose that changes in the capacity for reorganization of cytoskeletal elements induced by glucocorticoids are essential for efficient phagocytic uptake of apoptotic cells.

RAC1 regulates adherens junctions through endocytosis of E-cadherin.

The establishment of cadherin-dependent cell-cell contacts in human epidermal keratinocytes are known to be regulated by the Rac1 small GTP-binding protein, although the mechanisms by which Rac1 participates in the assembly or disruption of cell-cell adhesion are not well understood. In this study we utilized green fluorescent protein (GFP)-tagged Rac1 expression vectors to examine the subcellular distribution of Rac1 and its effects on E-cadherin-mediated cell-cell adhesion. Microinjection of keratinocytes with constitutively active Rac1 resulted in cell spreading and disruption of cell-cell contacts. The ability of Rac1 to disrupt cell-cell adhesion was dependent on colony size, with large established colonies being resistant to the effects of active Rac1. Disruption of cell-cell contacts in small preconfluent colonies was achieved through the selective recruitment of E-cadherin-catenin complexes to the perimeter of multiple large intracellular vesicles, which were bounded by GFP-tagged L61Rac1. Similar vesicles were observed in noninjected keratinocytes when cell-cell adhesion was disrupted by removal of extracellular calcium or with the use of an E-cadherin blocking antibody. Moreover, formation of these structures in noninjected keratinocytes was dependent on endogenous Rac1 activity. Expression of GFP-tagged effector mutants of Rac1 in keratinocytes demonstrated that reorganization of the actin cytoskeleton was important for vesicle formation. Characterization of these Rac1-induced vesicles revealed that they were endosomal in nature and tightly colocalized with the transferrin receptor, a marker for recycling endosomes. Expression of GFP-L61Rac1 inhibited uptake of transferrin-biotin, suggesting that the endocytosis of E-cadherin was a clathrin-independent mechanism. This was supported by the observation that caveolin, but not clathrin, localized around these structures. Furthermore, an inhibitory form of dynamin, known to inhibit internalization of caveolae, inhibited formation of cadherin vesicles. Our data suggest that Rac1 regulates adherens junctions via clathrin independent endocytosis of E-cadherin.

Co-localization of Rac1 and E-cadherin in human epidermal keratinocytes.

The Rac1 small GTP-binding protein is known to be involved in reorganization of the actin cytoskeleton and in regulation of intracellular signal transduction. The assembly and maintenance of cadherin-based cell cell junctions in epidermal keratinocytes is thought to be dependent on activity of Rac1. In this study we have generated green fluorescent protein (GFP)-tagged wild type, dominant negative and constitutively active Rac1 expression vectors and analyzed distribution of Rac1 following microinjection of human SCC12F epidermal keratinocytes. Wild type, dominant negative and constitutively active GFP Rac1 proteins distribute to sites of cell cell adhesion and co-localize with E-cadherin and the catenins. Disruption of cadherin-based junctions by reduction in extracellular calcium concentrations, or by use of antibodies to E-cadherin, results in redistribution of Rac1 away from sites of cell cell interaction but the co-localization with E-cadherin is maintained. In addition, expression of constitutively active GFP Rac1 results in formation of membrane ruffles on the apical surface of cells and intracellular vesicles. Interestingly, co-localization of Rac1 with E-cadherin is maintained in these structures. In contrast to previously published work we find that expression of dominant negative Rac1 neither disrupts cell cell adhesion nor prevents assembly of new cadherin-based adhesion structures.

Role of the cytoskeleton in rapid activation of CD11b/CD18 function and its subsequent downregulation in neutrophils.

When rolling adherent neutrophils are stimulated, they rapidly immobilize through activation of integrin CD11b/CD18, and then modulate attachment through this integrin to allow migration. We investigated links between cytoskeletal rearrangement and changes in function of integrin CD11b/CD18 in neutrophils stimulated with formyl peptide (fMLP). Neutrophils treated with the actin-polymerizing agent jasplakinolide became rolling adherent on monolayers of activated platelets, but could not use CD11b/CD18 to become immobilised when fMLP was perfused over them. If treated with jasplakinolide after fMLP, the cells stopped migrating but could not detach when fMLP was removed. Jasplakinolide did not inhibit changes in intracellular Ca(2+) seen after fMLP treatment, or inhibit neutrophil immobilisation induced by externally added Mn(2+). Thus cytoskeletal rearrangement was directly implicated in upregulation and, later, downregulation of CD11b/CD18 binding. Inhibition of RhoA with C3-transferase caused a dose-dependent reduction of initial rolling adhesion of neutrophils, and reduced the rate of migration after stimulation; however, neither the conversion of rolling to stationary adhesion, nor the ability of neutrophils to detach on removal of the stimulus, were inhibited. Thus, Rho may regulate actin polymerisation and motility in neutrophils, but did not appear to control integrin-mediated adhesion itself. Integrin binding may be promoted by disruption of links to the cytoskeleton, effected through depolymerisation of actin or cleavage of linking protein talin by calpain. Disruption of actin filaments with cytochalasin D did not, however, cause integrin-mediated immobilisation of rolling neutrophils. Although the calpain inhibitor calpeptin did inhibit the adhesion response to fMLP, this was only at doses where actin polymerisation was also ablated. We suggest that the cytoskeleton actively regulates binding conformation of CD11b/CD18 as well as its mobility in the membrane.

Visualizing muscle cell migration in situ.

BACKGROUND: Cell migration has been studied extensively by manipulating and observing cells bathed in putative chemotactic or chemokinetic agents on planar substrates. This environment differs from that in vivo and, consequently, the cells can behave abnormally. Embryo slices provide an optically accessible system for studying cellular navigation pathways during development. We extended this system to observe the migration of muscle precursors from the somite into the forelimb, their cellular morphology, and the localization of green fluorescent protein (GFP)-tagged adhesion-related molecules under normal and perturbed conditions. RESULTS: Muscle precursors initiated migration synchronously and migrated in broad, rather than highly defined, regions. Bursts of directed migration were followed by periods of meandering or extension and retraction of cell protrusions. Although paxillin did not localize to discernible intracellular structures, we found that alpha-actinin localized to linear, punctate structures, and the alpha5 integrin to some focal complexes and/or vesicle-like concentrations. Alterations in the expression of adhesion molecules inhibited migration. The muscle precursors migrating in situ formed unusually large, long-lived protrusions that were polarized in the direction of migration. Unlike wild-type Rac, a constitutively active Rac localized continuously around the cell surface and promoted random protrusive activity and migration. CONCLUSIONS: The observation of cellular migration and the dynamics of molecular organization at high temporal and spatial resolution in situ is feasible. Migration from the somite to the wing bud is discontinuous and not highly stereotyped. In situ, local activation of Rac appears to produce large protrusions, which in turn, leads to directed migration. Adhesion can also regulate migration.

Cell vacuolation induced by the VacA cytotoxin of Helicobacter pylori is regulated by the Rac1 GTPase.

Chronic gastric infection with the Gram-negative bacterium Helicobacter pylori is a major contributing factor in the development of duodenal ulcers and is believed to be a significant risk factor in the development of gastric tumors. The VacA cytotoxin of H. pylori is a 90-kDa secreted protein that forms trans-membrane ion channels. In epithelial cells, VacA activity is associated with the rapid formation of acidic vacuoles enriched for late endosomal and lysosomal markers. Rac1 is a member of the Rho family of small GTP-binding proteins that regulate reorganization of the actin cytoskeleton and intracellular signal transduction and are being shown increasingly to play a role in membrane trafficking events. In this study we report that: (i) green fluorescent-tagged Rac1 localizes around the perimeter of the vacuoles induced by VacA; (ii) expression of dominant negative Rac1 in epithelial cells inhibits vacuole formation; (iii) expression of constitutively active Rac1 potentiates the activity of VacA. Taken together, these data demonstrate a role for Rac1 in the regulation of VacA activity.

Differential activation of focal adhesion kinase, Rho and Rac by the ninth and tenth FIII domains of fibronectin.

Fibronectins are widely expressed extracellular matrix ligands that are essential for many biological processes. Fibronectin-induced signaling pathways are elicited in diverse cell types when specific integrin receptors bind to the ninth and tenth FIII domains, FIII9-10. Integrin-mediated signal transduction involves activation of signaling pathways of the growth factor-dependent Ras-related small GTP-binding proteins Rho and Rac, and phosphorylation of focal adhesion kinase. We have dissected the requirement of FIII9 and FIII10 for Rho and Rac activity and phosphorylation of focal adhesion kinase in BHK fibroblasts and Swiss 3T3 cells. We demonstrate that FIII10 supports cell attachment but does not induce phosphorylation of focal adhesion kinase. In Swiss 3T3 cells, growth factor-independent phosphorylation of focal adhesion kinase and downstream adhesion events are dependent upon the presence of FIII9 in the intact FIII9-10 pair, whereas FIII10-mediated focal adhesion kinase phosphorylation requires a synergistic signal from growth factors. Furthermore, FIII10 is able to elicit cellular responses mediated by Rho, but not Rac, whereas FIII9-10 can elicit both Rho- and Rac-mediated responses. We propose that activation of specific integrin subunits by the FIII10 and FIII9-10 ligands elicits distinct signaling events. This may represent a general molecular mechanism for activation of receptor-specific signaling pathways by a multi-domain ligand.

The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts.

Cadherins are calcium-dependent cell-cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell-cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell-cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell-cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell- cell contact formation and the remodelling of actin filaments in epithelial cells.

Regulation of the actin cytoskeleton, integrins and cell growth by the Rho family of small GTPases.

The Rho family of small GTP binding proteins play a key part in regulating the actin cytoskeleton and cell adhesion through integrin receptors. In addition, these proteins regulate signal transduction pathways essential for normal cell growth. Many of the molecules that regulate Rho have oncogenic activity, suggesting that members of the Rho family may have an important role in tumour formation.

The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases.

Interaction of cells with extracellular matrix via integrin adhesion receptors plays an important role in a wide range of cellular: functions, for example cell growth, movement, and differentiation. Upon interaction with substrate, integrins cluster and associate with a variety of cytoplasmic proteins to form focal complexes and with the actin cytoskeleton. Although the intracellular signals induced by integrins are at present undefined, it is thought that they are mediated by proteins recruited to the focal complexes. It has been suggested, for example, that after recruitment to focal adhesions p125FAK can activate the ERK1/2 MAP kinase cascade. We have previously reported that members of the rho family of small GTPases can trigger the assembly of focal complexes when activated in cells. Using microinjection techniques, we have now examined the role of the extracellular matrix and of the two GTP-binding proteins, rac and rho, in the assembly of integrin complexes in both mouse and human fibroblasts. We find that the interaction of integrins with extracellular matrix alone is not sufficient to induce integrin clustering and focal complex formation. Similarly, activation of rho or rac by extracellular growth factors does not lead to focal complex formation in the absence of matrix. Focal complexes are only assembled in the presence of both matrix and functionally active members of the rho family. In agreement with this, the interaction of integrins with matrix in the absence of rho/rac activity is unable to activate the ERK1/2 kinases in Swiss 3T3 cells. In fact, ERK1/2 can be activated fully by growth factors in the absence of matrix and it seems unlikely, therefore, that the adhesion dependence of fibroblast growth is mediated through the ras/MAP kinase pathway. We conclude that extracellular matrix is not sufficient to trigger focal complex assembly and subsequent integrin-dependent signal transduction in the absence of functionally active members of the rho family of GTPases.

Regulation of cell surface beta 1 integrin levels during keratinocyte terminal differentiation.

Integrins of the beta 1 family play a central role in controlling adhesion and terminal differentiation within the epidermis. When human epidermal keratinocytes undergo terminal differentiation, intracellular transport of newly synthesized integrins is inhibited, and mature receptors are lost from the cell surface. We have examined the mechanisms underlying these processes, using an experimental model in which keratinocytes are placed in suspension to induce terminal differentiation. The block in intracellular transport was keratinocyte- and integrin-specific since it was not observed when fibroblasts were placed in suspension and did not affect E-cadherin synthesis in suspended keratinocytes. Newly synthesized beta 1 integrins associated with an endoplasmic reticulum resident protein, calnexin; the association was prolonged when keratinocytes were placed in suspension, suggesting a role for calnexin in the inhibition of transport. After 24 h, the level of beta 1 integrin mRNA declines in suspended keratinocytes, reflecting inhibition of gene transcription, but in fibroblasts, the level remained constant. Transport of integrins could be blocked in both adherent keratinocytes and fibroblasts by inhibiting total protein synthesis, raising the possibility that transport is coupled to de novo integrin synthesis. The fate of receptors on the surface of keratinocytes was followed by confocal immunofluorescence microscopy, immunoelectron microscopy, and biochemical analysis: with the onset of terminal differentiation, endocytosed receptors were transported to the lysosomes. These experiments reveal novel mechanisms by which integrin levels can be controlled. Together with our earlier evidence for transcriptional regulation and affinity modulation of integrins, they highlight the complexity of the mechanisms which ensure that the onset of terminal differentiation is linked to detachment of keratinocytes from the underlying basement membrane.

Functional down-regulation of alpha 5 beta 1 integrin in keratinocytes is reversible but commitment to terminal differentiation is not.

Extracellular matrix receptors of the integrin family have a dual role in the epidermis, regulating both adhesion and differentiation. Loss of contact with the extracellular matrix causes keratinocytes to become committed to terminal differentiation, and results in a decrease in the ability of the alpha 5 beta 1 integrin to bind fibronectin. We have investigated whether the decrease in ligand-binding ability is reversible and, if so, whether commitment to terminal differentiation can also be reversed. Keratinocytes that had been placed in suspension for 5 hours to induce commitment were compared with the starting population (0 hour cells) in the presence or absence of 8A2, an activating anti-beta 1 antibody. 8A2 IgG or FAb fragments increased the amount of alpha 5 beta 1 in cell extracts that bound to fibronectin-Sepharose and in the presence of 8A2 the amount of bound alpha 5 beta 1 in 0 hour and 5 hour extracts was equal. 8A2 also restored alpha 5 beta 1 function in adhesion assays of intact 5 hour cells. Ca2+, Mg2+ and Mn2+ alone, at concentrations of up to 1 mM, did not increase the adhesiveness of 5 hour cells relative to 0 hour cells; however, the effect of 8A2 on keratinocytes was dependent on Ca2+. Although 8A2 restored alpha 5 beta 1 ligand-binding ability it did not prevent committed cells from withdrawing from the cell cycle and expressing involucrin, a differentiation marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions.

Suspension-induced terminal differentiation of human epidermal keratinocytes can be inhibited by fibronectin through binding to the alpha 5 beta 1 integrin. We have investigated the effect of fibronectin on expression of integrins and proteins of the actin cytoskeleton and have explored the nature of the differentiation stimulus by testing different combinations of anti-integrin monoclonal antibodies or extracellular matrix proteins in the suspension assay. Fibronectin prolonged cell surface expression of beta 1 integrins but did not overcome the inhibition of intracellular transport of integrins that occurs when keratinocytes are placed in suspension. Fibronectin did not prevent the suspension-induced decline in the level of mRNAs encoding the beta 1 integrin subunit, actin, filamin and alpha-actinin; furthermore, the inhibition of terminal differentiation did not depend on the state of assembly of microfilaments or microtubules. Terminal differentiation could be partially inhibited by an adhesion-blocking monoclonal antibody to the beta 1 integrin subunit or by a combination of adhesion blocking antibodies recognising the alpha subunits that associate with beta 1 (alpha 2, alpha 3 and alpha 5). Although laminin and type IV collagen do not inhibit terminal differentiation individually, they were inhibitory when added to cells in combination with a low concentration of fibronectin. We conclude that the proportion of keratinocyte beta 1 integrins occupied by ligand can regulate the initiation of terminal differentiation independently of the state of assembly of the actin cytoskeleton.

Analysis of the tumorigenic potential of common marmoset lymphoblastoid cells expressing a constitutively activated c-myc gene.

The respective roles of Epstein-Barr virus (EBV) and c-myc in the pathogenesis of endemic Burkitt's lymphoma (BL) are unclear. In order to help resolve the question whether constitutive expression of the c-myc gene in an EBV-immortalised B cell is sufficient to induce a tumorigenic phenotype, B cells from a common marmoset (Callithrix jacchus) were immortalised with EBV, transfected with a constitutively activated c-myc gene and inoculated into the host animals. Despite the cell line transfected with c-myc displaying enhanced growth characteristics, in vitro and in vivo experiments demonstrated that this was not sufficient to induce a tumorigenic phenotype. This supports our previous findings with EBV-immortalised human B cells transfected with an activated c-myc gene (Hotchin et al., 1990).

Transcriptional and post-translational regulation of beta 1 integrin expression during keratinocyte terminal differentiation.

During suspension-induced terminal differentiation of human epidermal keratinocytes, the alpha 5 beta 1 integrin is down-regulated in two stages: first, the ability of the receptor to bind fibronectin is reduced; later, the receptor is lost from the cell surface, and the level of the subunit mRNAs declines. We have begun to examine the mechanisms that regulate these events. Pulse-chase experiments showed that when keratinocytes were placed in suspension to induce terminal differentiation maturation of the beta 1 subunit and its associated alpha subunits was prevented. The inhibition of maturation was at the stage of N-linked glycosylation in the Golgi, because the immature integrin subunits were sensitive to endoglycosidase H digestion and the inhibition could be mimicked in adherent cells by treatment with 1-deoxymannojirimycin. In 1-deoxymannojirimycin-treated adherent keratinocytes, immature integrin subunits reached the cell surface; however, in keratinocytes induced to differentiate in suspension, no beta 1-integrin precursors were detected on the cell surface. Thus commitment to terminal differentiation results in a block both in integrin glycosylation and transport to the cell surface; down-regulation of receptor function must therefore involve modulation of pre-existing receptor on the cell surface. Although fibronectin or rabbit antiserum to alpha 5 beta 1 can inhibit suspension-induced terminal differentiation they did not overcome the inhibition of glycosylation. Nuclear run-on assays showed that transcription of the alpha 5 and beta 1 genes was switched off during suspension-induced terminal differentiation, and treatment of adherent keratinocytes with actinomycin D suggested that the half-lives of the alpha 5 and beta 1 mRNAs were similar in adherent and suspended cells. Thus, loss of alpha 5 beta 1 from the cell surface reflects both inhibition of transcription of the subunit genes and inhibition of maturation and intracellular transport of newly synthesized subunits.

A 19-year-old man with leucocyte adhesion deficiency. In vitro and in vivo studies of leucocyte function.

We describe a male patient with leucocyte adhesion molecule deficiency (LAD) of moderate phenotype. Although diagnosis was made only 2 years before his death, the patient survived until 19 years of age. This enabled us to perform a number of novel investigation, both in vivo and in vitro, relating to his leucocyte biology. Monocytes cultured in vitro matured into morphologically normal, phagocytically capable macrophages, which were able to recognize aged 'apoptotic' neutrophils. By injection of radiolabelled autologous neutrophils we demonstrated a prolonged neutrophil half-life, but normal margination, de-margination on exercise, and splenic pooling. Neutrophil adherence in vitro to vascular endothelium was normal. Histological examination of the patient's lungs at post-mortem showed intravascular aggregation of polymorphonuclear leucocytes but a paucity of cells in the interstitium and alveolar spaces. These findings indicate that the peripheral blood leucocytosis commonly observed in these patients may be due to prolonged intravascular neutrophil survival, and suggest that CD11/18 molecules have an important role in facilitating neutrophil emigration from blood vessels at sites of inflammation.