Monocytic origin and postnatal mitosis of intravascular macrophages in the porcine lung

Ultrastructural studies of near-term to 2-month-old pigs were done to document characteristics and developmental changes of intravascular macrophages in pulmonary capillaries. Evidence is presented that blood monocytes colonize the porcine lung perinatally, replicate within capillaries postnatally, and attach to endothelium by intercellular junctions during differentiation. Major ultrastructural features of differentiated intravascular macrophages are adhesion to capillary endothelium, pseudopods, phagosomes, and tubular structures of micropinocytosis vermiformis. Ultrastructure indicates that intravascular macrophages are cells of the mononuclear phagocyte system involved in several functions (eg, blood cell sequestration) that are usually attributed to hepatolineal macrophages. In newborn and 3-day-old pigs, the majority of cells closely apposed to endothelium consisted of few differentiated monocytes, but in 7-day-old and older animals, most cells that were joined to endothelium had characteristics of differentiated intravascular macrophages.     

Expression of intercellular adhesion molecule-1 in atherosclerotic plaques.

Immunohistochemistry of human atherosclerotic arteries demonstrates expression of the intercellular adhesion molecule-1 (ICAM-1) on endothelial cells, macrophages, and smooth muscle cells of the plaques. Normal arterial endothelial cells and intimal smooth muscle outside plaques give weaker or negative reactions; these differ from the strong endothelial expression in small vessels. Quantitative color-image analysis of the endothelial layer shows increased expression of ICAM-1 in all subtypes of atherosclerotic lesions, except fibrous plaques. Endothelial expression of ICAM-1 may be involved in the recruitment of monocytes to the lesion, as suggested by its role in the entry of leukocytes, including monocytes, into foci of inflammation. Collaboration with other mechanisms, particularly chemoattractant factors, may be important for this effect. ICAM-1 enhanced monocyte recruitment is a potential mechanism for the growth of an atherosclerotic plaque.     

Ultrastructural study of expression of adhesion molecules between blood monocytes and alveolar macrophages from patients with pulmonary sarcoidosis

Pulmonary sarcoidosis is a chronic inflammatory disorder characterized by the presence of activated T cells and alveolar macrophages at sites of inflammation. These cells are recovered from bronchoalveolar lavage (BAL) from sarcoid patients in order to evaluate the expression of various markers on cell surfaces that should determine the diagnosis in sarcoidosis. In this work we compared the expression of VLA-4, VLA-5, Mac-1, ICAM-1 and VCAM- 1 adhesion molecules, at ultrastructural level, between blood monocytes and alveolar macrophages obtained from BAL, from patients with pulmonary sarcoidosis. Cells obtained from blood and BAL were fixed, embedded in LRWhite and then ultrathin sections were incubated with monoclonal antibodies against VLA-4, VLA-5, Mac-1, ICAM-1 and VCAM-1. The results showed a more evident labelling of all adhesion molecules on alveolar macrophages when compared to blood monocytes. The labelling was seen at cell surface, at cytoplasm and small vacuoles. The differences on adhesion molecule distributions from blood monocytes to alveolar macrophages suggest that changes in the expression of these molecules occur during pulmonary inflammatory response. Lymphocytes from BAL or blood had a weak label for these molecules.     

Studies on the pathogenesis of atherosclerosis. I. Adhesion and emigration of mononuclear cells in the aorta of hypercholesterolemic rats.

In rats with diet-induced hypercholesterolemia, two concomitant changes began to occur within 1 week and persisted for 1 year: an increase in total plasma cholesterol and an increase in the number of mononuclear cells adhering to the aortic intima (up to values 50 times normal). Adherent cells were approximately 90% monocytes and approximately 10% lymphocytes. Adhesion was focal, with some preference for ostia of aortic branches; it was followed by migration into the subendothelial space. The subendothelial monocytes/macrophages progressively became foam cells, thus giving rise to microscopic "fatty streaks." Ultimately, typical atherosclerotic plaques were formed. Four possible mechanisms of increased cell adhesion are suggested. Endothelial changes were mild; myelin figures arising from the endothelial surface were seen by electron microscopy. Endothelial denudation was never observed, neither in light-microscopic preparations stained with AgNO3 nor by ultrastructure. Platelet participation was minimal. It is concluded that in this model atherosclerotic plaques are initiated by mononuclear cell adhesion and emigration; endothelial denudation is not a necessary step in their pathogenesis.     

The effects of monocytes on the transendothelial migration of T lymphocytes.

In vivo cell-mediated immune reactions are characterized by mixtures of monocytes and T cells. The purpose of this study was to investigate the role of monocytes on T-cell migration and induction of endothelial adhesion molecules. The in vitro model consisted of adding peripheral blood mononuclear cells (PBMC), T cells or mixtures of monocytes and T cells, to endothelial cells on a porous membrane and using flow cytometry to distinguish between the monocyte and lymphocyte components. PBMC and PBMC supernatants were highly potent at upregulating intercellular adhesion molecule-1 (ICAM-1) and inducing expression of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Induction by supernatants was inhibited by antibodies to tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL)-1 beta. Using monocyte-enriched populations, as few as one monocyte to 100 endothelial cells was sufficient to upregulate adhesion molecules. Fixed monocytes also induced adhesion molecules and expressed surface-bound cytokines. In contrast, highly purified unstimulated T cells were not found to induce adhesion molecules at 4, 6, 24 or 48 hr of coculture. Purified T cells showed low-level migration through resting (VCAM-1 negative) endothelium, which was approximately doubled by addition of small numbers of monocytes or TNF-alpha. In conclusion, monocytes, via cell surface or released cytokines play an essential role in allowing large-scale recruitment of T cells to inflammatory sites in vivo.     

Atheromatous plaque macrophages produce plasminogen activator inhibitor type-1 and stimulate its production by endothelial cells and vascular smooth muscle cells.

The capacity of macrophages to influence directly and indirectly fibrinolytic processes in atherosclerosis was studied using macrophages isolated from atherosclerotic plaques of patients undergoing surgical repair of distal aortic and femoral arteries. These cells were characterized by their morphology, adherence, esterase positivity, and expression of CD14 antigen. Production of plasminogen activator inhibitor type-1 (PAI-1) by plaque macrophages (6.7 +/- 2.7 ng/10(5) cells/24 hours [mean +/- SEM]) was significantly greater than PAI-1 production by blood monocytes isolated simultaneously from the same patients (1.8 +/- 1.5 ng/10(5) cells/24 hours). Production of tissue type plasminogen activator and urokinase type was not augmented compared to blood monocytes. Conditioned medium from cultured plaque macrophages significantly increased production of PAI-1 by endothelial cells (85 +/- 11% above basal) and vascular smooth muscle cells (25 +/- 10%) in vitro. This response was significantly greater than the response to monocyte-conditioned medium (endothelial cells 38 +/- 11%, vascular smooth muscle cells 2.5 +/- 2.0%). Stimulation of endothelial cell PAI-1 production by macrophage-conditioned medium was partially inhibitable by a monoclonal antibody to transforming growth factor-beta. Tissue type plasminogen activator production by endothelial cells and vascular smooth muscle cells was not affected by plaque macrophage- or monocyte-conditioned medium. Urokinase type plasminogen activator production by endothelial cells and vascular smooth muscle cells was undetectable in control medium and was augmented to similar levels in response to plaque macrophage- and monocyte-conditioned media. These results demonstrate upregulation of PAI-1 production by macrophages in atheromatous plaques and the capacity of soluble products from plaque macrophages to upregulate PAI-1 production by endothelial cells and vascular smooth muscle cells in vitro. These data suggest that macrophages in atherosclerotic plaques may inhibit thrombolysis both directly and indirectly by effects of their soluble products on endothelial cells and vascular smooth muscle cells.     

Monocyte migration across pulmonary membranes in mice infected with cytomegalovirus.

Ultrastructural evidence of monocyte migration across pulmonary membranes was found in immunosuppressed mice infected with murine cytomegalovirus (CMV). A previous study showed that monocytes are the only cells that contain virus particles in this experimental model. These cells migrate across the vascular endothelium and into the interstitial space. They then move through the basement membranes of alveoli and bronchioles into newly formed spaces between the basement membrane and the epithelium. Concurrently, zonulae adhaerentes develop between the monocytes and the investing epithelial cells. Gaps then appear to develop in the epithelium, probably at intercellular junctions, and the monocytes migrate into the air spaces. These morphologic observations corroborate earlier evidence which suggested that monocytes are the precursors of alveolar macrophages.     

Increase in the adhesion molecule P-selectin in endothelium overlying atherosclerotic plaques. Coexpression with intercellular adhesion molecule-1.

P-selectin (GMP-140) is an adhesion molecule present within endothelial cells that is rapidly translocated to the cell membrane upon activation, where it mediates endothelial-leukocyte interactions. Immunohistochemical analysis of human atherosclerotic plaques has shown strong expression of P-selectin by the endothelium overlying active atherosclerotic plaques. P-selectin is not, however, detected in normal arterial endothelium or in endothelium overlying inactive fibrous plaques. Color image analysis was used to quantitate the degree of P-selectin expression in the endothelium and demonstrates a statistically significant increase in P-selectin expression by atherosclerotic endothelial cells. Double immunofluorescence shows that some of this P-selectin is expressed on the luminal surface of the endothelial cells. Previous work has demonstrated a significant up-regulation in the expression of the intercellular adhesion molecule-1 in atherosclerotic endothelium and a study on the expression of intercellular adhesion molecule-1 and P-selectin in atherosclerosis shows a highly positive correlation. These results suggest that the selective and cooperative expression of P-selectin and intercellular adhesion molecule-1 may be involved in the recruitment of monocytes into sites of atherosclerosis.     

Atherosclerotic lesions grow through recruitment and proliferation of circulating monocytes in a murine model

Macrophage-derived foam cells in developing atherosclerotic lesions may potentially originate either from recruitment of circulating monocytes or from migration of resident tissue macrophages. In this study, we have determined the source of intimal macrophages in the apoE-knockout mouse flow-cessation/hypercholesterolemia model of atherosclerosis using a bone marrow transplantation approach. We also examined the time course and spatial distribution of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression to assess whether endothelial adhesion molecules were involved in recruitment of either circulating monocytes or resident macrophages. We used allelic variants of the mouse common leukocyte antigen (CD45) to distinguish host-derived and donor-derived white blood cells (WBCs) both in blood and in macrophage-rich carotid lesions. We found that the distribution of CD45 isoforms in lesions is similar to that of circulating WBCs, whereas the host-type CD45 isoform is more prevalent in resident adventitial macrophages. These data indicate that macrophage-derived foam cells in the lesion derive mainly from circulating precursors rather than from resident macrophages. The corresponding time course of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression suggests that recruitment of circulating WBCs by endothelial adhesion molecules is likely to be more important during lesion initiation than during the later phase of rapid lesion growth.     

Chlamydia pneumoniae infection increases adherence of mouse macrophages to mouse endothelial cells in vitro and to aortas ex vivo

Interactions between monocytes/macrophages and endothelial cells play an important role in the pathogenesis of atherosclerosis, and the adherence of monocytes to the arterial endothelium is one of the early events in atherogenesis. In the present study, peritoneal macrophages harvested from green fluorescent protein (GFP) transgenic mice were used to analyze how Chlamydia pneumoniae infection affects the adherence of GFP-macrophages to mouse endothelial cells in vitro and to the aorta from normolipidemic and hyperlipidemic mice ex vivo. In vitro studies showed that C. pneumoniae-infected GFP-macrophages adhered better than uninfected macrophages to endothelial cells and GFP-macrophages adhered better to infected than uninfected endothelial cells. The ex vivo studies showed that C. pneumoniae-infected macrophages adhered better than uninfected macrophages to aortas from both normolipidemic and hyperlipidemic C57BL/6J mice and apolipoprotein E (ApoE)-deficient mice. In contrast, adherence of C. pneumoniae-infected macrophages to the aortas of intercellular adhesion molecule 1 (ICAM-1) knockout mice was not enhanced, suggesting that ICAM-1 is crucial for activation of the adherence of C. pneumoniae-infected macrophages to the endothelium. In conclusion, the present study defined a homing mechanism by which C. pneumoniae promotes the adherence of mononuclear phagocytes to the endothelium at the site of atherosclerotic lesion formation to promote the progression of atherosclerosis.     

Leukocytes in chemotactic-fragment-induced lung inflammation. Vascular emigration and alveolar surface migration.

Lung inflammation was induced in rabbits by intratracheal injections of chemotactic fragments obtained from zymosan-activated serum (CF-ZAS), and the route of vascular emigration and alveolar surface interaction of polymorphonuclear leukocytes (PMNs) and monocytes migrating into the lung was characterized by transmission (TEM) and scanning (SEM) electron-microscopic examination. Leukocytes migrated from capillaries and venules into the alveolar wall interstitium by adherence to the vascular endothelium and migration through the endothelial intracellular junction to attain a position between a reapposed endothelial cell junction and the vascular basement membrane. The cells then migrated into the interstitium through a narrow opening in the basement membrane. Leukocyte entrance into the alveolar space from the interstitium appeared to occur through small openings in the epithelial basement membrane at or near the Type I epithelial intercellular junction. Once in the alveolus, PMNs and macrophages demonstrated surface adherence and spreading along with evidence of migration, pseudopod extension, interalveolar pore transit, and retraction fiber formation. This study indicates the leukocyte influx into the alveolus in acute chemotactic-factor-induced inflammation is via a continuum of migrational activity, beginning at the pulmonary capillary endothelial surface and persisting on the alveolar epithelial surface.     

Macrophage tropism of rabbit hemorrhagic disease virus is associated with vascular pathology

To delineate the interactions between rabbit hemorrhagic disease virus (RHDV) and host cells, organ and cellular targets of infection were identified in vivo. Viral specific antigens were detected by immunohistochemistry in liver, lung, spleen and lymph nodes cells. Also, intravascular infected cells were detected in most organs including kidneys, myocardium, thymus and central nervous system. To further characterize infected target cells, viral proteins and cell-specific surface antigens were identified simultaneously in double labeling experiments. Numerous lymphoid organ macrophages, from the splenic red pulp, circulating monocytes, alveolar macrophages and Kupffer cells were double labeled, demonstrating that cells of the mononuclear phagocyte lineage are major hosts for RHDV. Double labeling for other specific cell markers were negative. The distribution of viral antigens in these tissues coincided with those areas where cells presented morphology of apoptosis. Association of intravascular monocyte infection and apoptosis, could represent a possible mechanism to develop disseminated intravascular coagulation.     

Cell type-specific mRNA quantitation in non-neoplastic tissues after laser-assisted cell picking.

OBJECTIVE: Cell type-specific mRNA quantitation can be reliably performed after harvesting less than 20 cell profiles from haemalaun-stained cryosections by laser-assisted cell picking. Up to now it has been unclear to what extent these techniques can be used to analyze differential gene expression in complex tissues. METHODS: Using a rat model of experimental endotoxin priming of the lung various pulmonary cell types were microdissected from isolated perfused and ventilated rat lungs after aerosol lipopolysaccharide/interferon-gamma stimulation. RESULTS: Porphobilinogen deaminase housekeeping gene (PBGD) and nitric oxide synthase II (NOSII) mRNA in arterial endothelial cells (AEC), bronchiolar epithelial cells (BEC), alveolar septum containing monocytes/macrophages (AS+), alveolar septum without monocytes/macrophages (AS-) and intraluminar alveolar macrophages (AM) could be quantified by real-time RT-PCR. The strongest upregulation of NOSII mRNA occurred in AM, while minimal NOSII expression was detected in BEC, AS+ and AS-. In AEC NOSII mRNA was not detectable. CONCLUSION: The combination of laser microdissection and real-time RT-PCR is a valuable tool for the quantitative in situ characterization of differential gene expression within complex tissues.     

Localized adhesion of monocytes to human atherosclerotic plaques demonstrated in vitro: implications for atherogenesis.

Blood-derived macrophages in the arterial intima are a characteristic feature of active atherosclerotic plaques. Adherent monocytes on the luminal surface and increased adhesion molecules on the endothelium have suggested that specific molecular mechanisms are involved in monocyte/macrophage traffic into the arterial wall. Adhesion of human monocytes and related cell lines was therefore studied in vitro to histological sections of human plaques. At 37 degrees C, these cells bound selectively to the plaques. Binding to the endothelium occurred and was also present extensively in the diseased intima. Inhibition studies showed that the endothelial and general intimal binding had largely similar molecular properties. Strong inhibition was produced by antibodies to the monocyte-specific adhesion molecule CD14, to beta2 integrins, and to ICAM-1. Likewise, a peptide containing the Arg-Gly-Asp sequence was strongly inhibitory, suggesting that binding of leukocyte integrins to arterial extracellular matrix was synergistic with cell-cell interactions. A P-selectin antibody was exceptional in giving selective inhibition of endothelial adhesion, which correlates with the specific endothelial localization of this adhesion molecule. These results show that monocytes adhere to atherosclerotic plaques through the focal activation of multiple arterial wall adhesion molecules, confirming the adhesion hypothesis. A positive feedback theory for the pathogenesis of atherosclerosis can be suggested, based on the ability of macrophages in the wall to activate the endothelium, induce adhesion molecules, and facilitate additional monocyte entry. The adhesion assay provides a means for the identification of adhesion inhibitors with therapeutic potential.     

Immunohistochemical expression of MPO,CD163 and VEGF in inflammatory cells in acute respiratory distress syndrome: a case report

Acute respiratory distress syndrome (ARDS) is a serious medical condition occurring in patients with polytrauma, pulmonary or non-pulmonary sepsis, pneumonia and many other circumstances. It causes inflammation of the lung parenchyma leading to impaired gas exchange with a systemic release of inflammatory mediators, causing consequential lung tissue injury, hypoxemia and frequently multiple organ failure. The aim of current study was to describe expression of inflammatory markers (myeloperoxidase, CD163 and vascular endothelial growth factor) by the cells in acute phase of ARDS. The lung samples of a 20-year-old man who had suffered a serious motorbike accident were obtained for histological examination. He died on the seventh day as a consequence of respiratory failure. Our results imply that expression of CD163 was restricted to activated alveolar macrophages and monocytes. Immunopositivityof MPO was observed in neutrophil granulocytes within lung alveoli and lung blood vessels. Myeloperoxidase positivity was observed in alveolar macrophages, too. Vascular endothelial growth factor was expressed in cytoplasm of neutrophil granulocytes, monocytes, small-sized alveolar macrophages and type II pneumocytes localized mostly inside lung alveoli. On the contrary, no positivity was observed in lung endothelial cells of blood vessels.     

Production of tumor necrosis factor and interleukin-1 by macrophages from human atheromatous plaques.

The production of cytokines by atheromatous plaque macrophages from human endarterectomy tissue was assessed in vitro by short-term cell culture and in situ by immunohistology. Macrophages were isolated from plaques of 14 patients undergoing carotid endarterectomy and 7 patients undergoing reconstructive procedures on atheromatous distal aortic and femoral arteries. Tumor necrosis factor (TNF) and interleukin 1 (IL-1) production by plaque macrophages and blood monocytes isolated concurrently from these patients was assessed. TNF release by macrophages from carotid plaques (0.39 +/- 0.12 ng/10(6) cells/24 hours) was significantly augmented compared to the release by corresponding blood monocytes (0.014 +/- 0.011 ng/10(6) cells/24 hours, P = 0.03), and by macrophages from noncarotid lesions (0.038 +/- 0.036 ng/10(6) cells/24 hours, P < 0.04). Cellular TNF expression by macrophages within carotid plaques was also more prominent than in noncarotid lesions. By contrast, IL-1 production by plaque macrophages from both carotid and noncarotid plaques was not augmented compared to blood monocytes, and only infrequent and low-intensity labeling for IL-1 was present on macrophages within plaques from either group. These results provide functional and immunohistological evidence for increased production of TNF but not IL-1 by activated macrophages, indicating local and selective augmentation of cytokine production within carotid plaques. This suggests that macrophages play an active role in the inflammatory response within atheromatous carotid plaques.     

Comparison of arachidonic acid metabolism by pulmonary intravascular and alveolar macrophages exposed to particulate and soluble stimuli

Pulmonary intravascular macrophages, as prominent components of the pulmonary mononuclear phagocyte system, could be significant mediators of lung inflammation. We have shown that intravascular and alveolar macrophages metabolize exogenous arachidonic acid to its inflammatory metabolites via the lipoxygenase and cyclooxygenase pathways after exposure to the calcium ionophore A23187. In this study, we compare the metabolism of endogenous arachidonic acid by porcine intravascular and alveolar macrophages after exposure to soluble and particulate stimuli. Since intravascular and alveolar macrophages are exposed to various stimuli in vivo, it is essential to know the range of inflammatory mediators that these cells can generate. Alveolar macrophages attached to plastic and exposed to the various stimuli produced prostaglandin F2 alpha, 12-hydroxyheptade-catrienoic acid (HHT), hydroxyeicosatetraenoic acids (HETE), and leukotriene B4. In contrast, adherent and stimulated intravascular macrophages produced several cyclooxygenase products and lipoxygenase products including 5-HETE, 12-HETE, and leukotriene B4. Both macrophages released large amounts of arachidonic acid upon exposure to each stimulant. Intravascular macrophages that were adherent to plastic or were stimulated with glass, asbestos, or A23187 released significantly (p less than 0.05) more metabolized arachidonic acid than similarly treated alveolar macrophages. The major cyclooxygenase metabolite released by alveolar macrophages was prostaglandin 2 alpha, whereas HHT was the primary metabolite of intravascular macrophages. The major lipoxygenase metabolite released by both macrophage types was 5-HETE, but intravascular macrophages also released substantial amounts of 12-HETE and leukotriene B4. In both macrophage preparations, lipoxygenase products composed most released metabolites. After exposure to iron, asbestos, and A23187 intravascular macrophages released significantly more (p less than 0.05) lipoxygenase metabolites than alveolar macrophages. However, in alveolar macrophages, chrysotile asbestos induced greater activity by the cyclooxygenase pathway than by the lipoxygenase pathway. Both asbestos and iron spheres induced release of arachidonic acid and its metabolites, but the most potent stimulants in both macrophage preparations were A23187, zymosan, and lipopolysaccharide. We conclude that stimulated intravascular macrophages use both cyclooxygenase and lipoxygenase pathways to metabolize endogenous arachidonic acid, that these macrophages are metabolically more active than alveolar macrophages, and that both macrophage types are induced to metabolize arachidonic acid by various particulate and soluble stimuli. Furthermore, we have shown that intravascular macrophages predominantly utilize the lipoxygenase rather than cyclooxygenase pathways to metabolize endogenous arachidonic acid.     

Comparative O2-. responses of lung macrophages and blood phagocytic cells in the rat. Possible relevance to IgA immune complex induced lung injury

IgA immune complex-induced lung injury in the rat is oxygen radical mediated and partially complement-dependent but develops fully after neutrophil depletion. The extent to which monocytes, lung interstitial macrophages, and alveolar macrophages may be involved in the development of lung injury in this model is unclear. To further understand the pathogenesis of IgA lung injury, we have examined the capacity of phagocytic cells isolated from different anatomic compartments of the lung to produce toxic oxygen-derived metabolites. [3H]Thymidine pulse labeling and autoradiography as well as in vivo phagocytosis studies were used to distinguish macrophages isolated from the alveolar and interstitial compartments. Lung interstitial macrophages were characterized ultrastructurally, cytochemically, and functionally. Interstitial macrophages were relatively uniform in size, had blunt pseudopodia, and contained almost no intracytoplasmic lamellar inclusions compared to alveolar macrophages. Similar to monocytes and alveolar macrophages, interstitial macrophages contained nonspecific esterase activity and exhibited the capacity to phagocytize latex and opsonized zymosan particles. Lung interstitial and alveolar macrophages incubated with IgA immune complexes, IgG immune complexes, or phorbol ester (PMA) produced similar amounts of O2-. in a dose-dependent manner. In contrast, peripheral blood neutrophils responded to IgG immune complexes and PMA but not to IgA immune complexes. Monocytes produced a small amount of O2-. in response to PMA but almost no O2-. in response to IgA or IgG immune complexes. These data are consistent with recent in vivo studies which indicate that IgA immune complex lung injury is neutrophil independent. The data provide direct in vitro evidence that lung interstitial and alveolar macrophages produce O2-. following incubation with PMA, IgA, or IgG immune complexes and may therefore contribute to the development of oxygen radical mediated lung injury.     

Identification of differential protein expression associated with development of unstable human carotid plaques

Rupture-prone unstable arterial plaques develop concomitantly with the appearance of intraplaque hemorrhage and tissue ulceration, in association with deregulation of smooth muscle cell mitogenesis and leakage of newly formed blood vessels. Using microarray technology, we have identified novel protein deregulation associated with unstable carotid plaque regions. Overexpression of proapoptotic proteins caspase-9 and TRAF4 was seen in endothelial cells and smooth muscle cells from unstable hemorrhagic and ulcerated plaque regions. Topoisomerase-II-alpha (TOPO-II-alpha), which is associated with DNA repair mechanisms, was also overexpressed by these cells. Cell signaling molecules c-src, G-protein-coupled receptor kinase-interacting protein (GIT1), and c-jun N-terminal kinase (JNK) were up-regulated in endothelial cells from the same areas, whereas an increase in expression of junctional adhesion molecule-1 (JAM-1) in blood vessels and infiltrating macrophages from inflammatory regions might form part of a leukocyte rolling response, increasing the plaque volume. Grb2-like adaptor protein (Gads), responsible for differentiation of monocytes into macrophages, was expressed by macrophages from unstable plaques, suggesting a potential mechanism through which increased scavenging could occur in rupture-prone areas. We conclude that modulation of novel cell signaling intermediates, such as those described here, could be useful in the therapy of angiogenesis and apoptosis, designed to reduce unstable plaque formation.     

Differential function of LFA-1 family molecules (CD11 and CD18) in adhesion of human monocytes to melanoma and endothelial cells.

Human peripheral blood monocytes from normal, healthy donors express the leucocyte function-associated antigen (LFA)-1, CR3 and p150,95. These heterodimeric antigens are members of a glycoprotein family sharing a common beta subunit but endowed with distinct alpha chains. They have been shown to play an important role in cell-cell interactions. In the present study we have investigated the role of these molecules in the interaction of monocytes with endothelial cells and melanoma (tumour) cells. Heterotypic cell-cell interactions were studied in single cell conjugate assays and by adhesion of monocytes to monolayers of cells. The results demonstrate that monoclonal antibodies directed against LFA-1 alpha, CR3 alpha, p150,95 alpha and the common beta chain strongly reduce the number of conjugates (71, 50, 60 and 89% inhibition, respectively), formed between monocytes and melanoma or endothelial cells in a single cell assay. In contrast, adhesion of monocytes to monolayers of the same cells seems only to depend on p150,95, since only antibodies directed to the alpha chain of this molecule and to the common beta chain inhibited adhesion. Interestingly, the number of conjugates formed with melanoma cells in single cell assays was at least twice the number of conjugates formed between monocytes and endothelial cells, whereas no differences were observed in the adhesion of monocytes to monolayers of these cells. However, the basis for this phenomenon is not yet clear. These results indicate that not only LFA-1 but also CR3 and p150,95 can mediate adhesion to target cells in suspension, but that monocyte adhesion to monolayers is caused by a different mechanism in which the p150,95 molecule seems to play a prominent role.