Leukocyte- and platelet-derived microvesicle interactions following in vitro and in vivo activation of toll-like receptor 4 by lipopolysaccharide

Background

Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS).

Methodology/Principal Findings

Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens.

Conclusions/Significance

Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood.     

Tumour-associated antigens in culture medium of malignant melanoma cell strains

Summary The presence of melanoma-associated antigens naturally shed from cultured melanoma cells in spent culture medium was investigated by means of a leukocyte migration test and culture medium from four melanoma and two control cell strains.Leukocytes from 29/64 melanoma patients showed a positive reaction with spent culture medium from at least one melanoma cell strain, whereas leukocytes from only 4/25 patients with other cancers and 1/30 normal donors reacted. On the other hand, leukocytes from only 8/51 melanoma patients reacted with control culture medium. Only melanoma patients' leukocytes reacted with two or more of the melanoma cell strains used. Culture media from two melanoma cell strains were more reactive (25.3% and 29.4% positive tests with melanoma patients' leukocytes) than others (12.5% and 17.2% positive tests); this may represent either a qualitative difference (i.e., different antigens) or a quantitative one (i.e., different levels of antigen expression according to tissue culture conditions). Both inhibition and stimulation of migration were observed, but with one exception, on a given occasion, leukocytes from the same donor always reacted in the same way (i.e., either inhibition or stimulation). Migration stimulation was observed mainly with melanoma patients' leukocytes, and more especially when leukocytes were sampled from patients within a few weeks from tumour removal; migration stimulation may thus reflect a particular state of sensitization in patients.From the evidence obtained in these studies, it is concluded that spent culture medium from melanoma cell strains contains melanoma-associated antigen (s) that is (are) reactive in the leukocyte migration test and that this may contribute to the study of specific antitumour reactivity in patients and to the study and purification of tumour-associated antigens by providing an homogeneous source of antigens spontaneously released from tumour cells in conditions close to natural ones.     

Regulation of leukocyte glass adherence and tube leukocyte adherence inhibition (LAI) reactivity by serum factors in dogs with progressing or spontaneously regressing canine transmissible venereal sarcoma (CTVS)

Summary We examined the regulation of leukocyte glass adherence and tube leukocyte adherence inhibition (LAI) reactivity by serum factors in dogs with regressing or progressing canine transmissible venereal sarcomas (CTVS). Both regressor and progressor peripheral blood leukocytes (PBL), draining and nondraining lymph node cells (LNC), and splenic leukocytes were significantly responsive to CTVS antigen extract in tube LAI. In contrast, a significant decrease in basal glass adherence of progressor PBL, draining and nondraining LNC, and splenic leukocytes was observed. Normal glass adherence was restored to progressor leukocytes by extensive washing with warm serum-free media, while significant tube LAI responsiveness to CTVS antigen extract was maintained. Preincubation of regressor PBL and LNC with progressor sera in two-stage tube LAI decreased the basal glass adherence of treated leukocytes. This effect of progressor sera was heat labile, a characteristic of CTVS antigen. Collectively, these findings suggest that progessor leukocytes and progressor sera treated regressor leukocytes were activated by interaction with serum CTVS antigen and thus behaved in tube LAI as stimulated cells, even in the absence of CTVS antigen. Regressor but not progressor sera were shown to contain anti-CTVS IgG with specific arming activity for normal dog PBL, but not LNC in two-stage tube LAI. The nonadherent response of peripheral blood neutrophils in two-stage tube LAI was proportional to the concentration of arming IgG, whereas no change was observed in glass adherence of PBL. The results of this study define the role of progressor and regressor serum factors in the mechanism of tube LAI and demonstrate a relationship between leukocyte glass adherence and the clinical course of CTVS. These findings show that tube LAI is a simple and reproducible measure of active factors in the immune response to a tumor.This investigation was supported in part by grant, CA-23469, from the National Cancer Institute, DHHS, and is submitted as Scientific Contribution No. 1051, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, CT 06268 USA     

Detection of a factor suppressing leukocyte migration in the sera of allergy patients following antigen administration]

The effect on donor leukocyte migration of serum obtained from the patients with tuberculosis of the lungs, chronic pneumonia and healthy persons was studied after subcutaneous or intradermal injection of the microbial antigen (PPD, streptococcus and staphylococcus antigen). A factor inhibiting donor leukocyte migration appeared in the blood serum of sensitized individuals after the antigen injection. This factor proved to be localized in the serum fraction III obtained after the gel-filtration of sephadex G-200, and is sorbed by leukocytes.     

Modulation of the human in vitro antibody response by human leukocyte interferon preparations

The ability of human leukocyte Interferon to modulate the plaque-forming-cell response of human peripheral blood leukocytes to horse red blood cells was examined. Human peripheral blood mononuclear cells were cultured in vitro with the addition of varying doses of human leukocyte interferon 24 hr prior to, simultaneously with, and 24 hr after sensitization of the cultures with horse red blood cells. Plaque-forming-cell responses were measured 5 days after sensitization with antigen using poly-L-lysine-coupled horse red blood cell monolayers. When human leukocyte interferon preparations were added 24 hr prior to sensitization with antigen, a significant enhancement of the plaque-forming-cell response was observed. When the interferon was added simultaneously with antigen, the plaque-forming-cell response was significantly suppressed. Therefore, human leukocyte interferon appears to have a time-dependent immunomodulatory activity. The kinetics of immunomodulation appear to be different from those of previously described mouse models.     

Antigen-independent leukocyte random locomotion inhibitory activity in human ultrafiltrated leukocyte extracts

Human ultrafiltrated leukocyte extracts (MW < 5000) were fractionated by Sephadex G-10 column chromatography and the effects of these fractions on leukocyte random locomotion were investigated in vitro. Fr-4, one of these fractions, had significant leukocyte random locomotion inhibitory activity, independent of the presence of mononuclear leukocytes. This inhibitory activity was not due to cytotoxic effects on leukocytes. As seen by scanning electron microscopy, the number of cell surface pseudopods on leukocytes incubated with Fr-4 was reduced. Fr-4A, one of three fractions separated from Fr-4 by Sephadex G-25 column chromatography, significantly inhibited leukocyte random locomotion. Fr-4A contained numerous components, one of which was identified as 2-deoxyribose, on the basis of thin-layer chromatography. Biologically 2-deoxyribose showed an inhibitory effect on leukocyte locomotion and a reduction of the extrusion of pseudopods on the surface of leukocytes, at the range of assayed concentrations. This inhibitory activity is probably derived from 2-deoxyribose.     

Leukocyte-endothelial cell recognition: evidence of a common molecular mechanism shared by neutrophils, lymphocytes, and other leukocytes

The interaction of leukocytes with endothelial cells is intrinsic to the process of leukocyte extravasation, whether during the entry of blood polymorphonuclear leukocytes and monocytes into sites of acute and chronic inflammation, or during the homing of lymphocytes to lymphoid organs. A lymphocyte surface glycoprotein, defined by monoclonal antibody MEL-14, has been described that appears to mediate lymphocyte recognition of postcapillary venules in peripheral lymph nodes, and to control the migration of lymphocytes from the blood into these lymphoid organs. We now report that the antigenic determinant recognized by MEL-14 is present at high levels on other leukocytes as well, including neutrophils, monocytes, and eosinophils; and we demonstrate involvement of the MEL-14 antigen in neutrophil-endothelial cell interactions. MEL-14 immunoprecipitates a neutrophil surface protein of Mr approximately 100,000, similar in m.w. to the 80,000 to 90,000 dalton lymphocyte surface MEL-14 antigen, and it blocks the interaction of neutrophils with endothelial cells in an in vitro model of adhesion to postcapillary venules in lymph node frozen sections. Neutrophil binding to lymph node venules is also inhibited by PPME, a mannose-6-phosphate-rich yeast polysaccharide that is thought to mimic the endothelial cell ligand for the MEL-14-defined lymphocyte receptor. Interestingly, neither MEL-14 nor PPME exhibit a major effect on neutrophil binding to postcapillary venules in Peyer's patches, suggesting that as for lymphocytes, the neutrophil MEL-14 antigen is involved in recognition of tissue-specific endothelial determinants. Finally, we show that MEL-14 inhibits the capacity of neutrophils to migrate from the blood into sites of acute inflammation in the skin. These observations lead us to propose that receptors for tissue-specific endothelial determinants are utilized by neutrophils and lymphocytes and probably other leukocytes during the physiologic process of leukocyte extravasation in vivo.     

Alterations of polymorphonuclear leukocyte surface charge by stimulated lymphocyte supernatants

The effects of human lymphokines on the surface charge density of human polymorphonuclear (PMN) leukocytes have been determined using the laser Doppler technique of electrophoretic light scattering. Unfractionated antigen (streptokinase-streptodornase or candida)-stimulated lymphocyte supernatants were found to decrease the mode electrophoretic mobility by 14%. In order to identify the responsible factor, we subjected supernatants from concanavalin A-stimulated lymphocytes to gel filtration on Sephadex G-100 columns and assayed the fractions for their ability to alter PMN electrophoretic mobilities. Two distinct species in the molecular weight ranges of 30–60K and 10–20K, respectively, were found to decrease the electrophoretic mobilities of PMN leukocytes. We have observed no effect of leukocyte inhibitory factor (LIF) on the electrophoretic mobility distribution of PMN leukocytes over a varying period of time (0–8 hr) and over a range of 2- to 10-fold supernatant concentration. Pretreatment of PMN leukocytes with neuraminidase substantially reduced their electrophoretic mobility; the addition of LIF to these pretreated cells did not alter their electrophoretic mobility distribution further. The latter finding is particularly significant in view of the fact that neuraminidase pretreatment of the target cells is known to potentiate LIF activity. We conclude that the mechanism of the inhibition of leukocyte migration by LIF does not involve an alteration of the leukocyte surface charge density.     

Biochemical and MALDI analysis of the human sperm antigen gp20, homologue of leukocyte CD52.

In previous work we demonstrated that gp20, a sialoglycoprotein of human sperm is homologous to the leukocyte antigen CD52 and that anti-gp20 recognizes an antigen of the same molecular weight as that recognized by CAMPATH-1 (anti CD52) in leukocytes and sperm, but with some differences. In this study we used anti-gp20 to perform immunoblot analysis of many different sperm, seminal plasma and leukocyte samples. The sperm and seminal plasma antigens were similar and appeared to consist of two components, whereas the leukocyte antigen is unique. Evidence of the presence of two components of the sperm antigen, running respectively at about 19 and 21 kDa, was obtained by analyzing the purified antigen stained with Coomassie brilliant blue and by immunoblot analysis of the antigen after two-dimensional electrophoresis. Both components had an isoelectric point (pI) between 3 and 6. MALDI analysis of the purified antigen confirmed the presence of two components and indicated masses (Mr) of 8243 and 10908. The possible relationship between these findings and the presence of two forms of the CD52 gene differing at two aminoacids C-terminal to the GPI-anchor site has been discussed.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Leukocyte migration inhibition studies with Epstein-Barr virus (EBV) determined nuclear antigen (EBNA) in relation to the EBV-carrier status of the donor

Cell extracts from EBV-genome-carrying cell lines inhibit the migration of leukocytes from EBV-positive but not seronegative healthy donors. In the present study extracts from EBV-negative lines and their own in vitro EBV-converted sublines were used to induce migration inhibition with leukocytes from seronegative and seropositive individuals. A clear difference was found between the extracts from EBV-negative and positive cell lines. Significant migration inhibition could be obtained with antigen(s) associated with the virus nonproducer state. Since EBNA is known to be expressed by all nonproducer EBV-genome-carrying cells, we have compared the effect of partially purified EBNA and correspondingly prepared mock-EBNA on the leukocyte migration. Purified EBNA inhibited the leukocyte migration of EBV seropositives, whereas mock-EBNA had no such effect.     

Antibody formation by one or both lymphoid populations present in chimeric marmosets

Development of an isoimmune serum capable of identifying a specific leukocyte antigen in the marmoset, Saguinus fuscicollis illigeri, permitted detection of lymphoid cell chimerism in this species by the cytotoxic test. This reagent was then used to identify the cell population in the chimera responsible for antibody production against a test antigen, sheep red blood cells. Primary in vitro antibody formation as measured by plaque-forming cells with blood leukocytes or splenic lymphocytes of six animals chimeric for the leukocyte antigen, MLA-1, revealed an immune response by both cell types of the chimeric population from three animals and a response by only one cell type in the other three.     

Graphical representations of the class I MHC cleft

We describe computer graphics and computer aided manufacture of three-dimensional models designed specifically to elucidate the cleft in the class I human leukocyte antigen. The models evolve from computer graphical representations and provide a geometrically and chemically concise and detailed view of the antigen binding site. The techniques provide a new approach to representations of binding sites. The model provides sufficient detail to support binding specificities analysis of active sites involved in protein and DNA binding.     

Expression by human fetal organs of organ-specific cancer neoantigens as measured by leukocyte adherence inhibition

Summary Human cancers express organ-specific cancer neoantigens (OSN) as determined by in vitro leukocyte responses to extracts of cancers by the tumor host. In this study, we determined whether the OSNs were normal developmental proteins that were expressed by fetal organs and re-expressed with oncogenesis. Fetal extracts, principally of lung and colon but also of liver and kidney, were tested for their ability to induce leukocyte adherence inhibition (LAI) as compared to extracts from adult tissues of the same organ. Leukocytes from lung cancer patients showed positive LAI responses to 13- and 19-week fetal lung tissue. Likewise, leukocytes from colon cancer patients showed positive LAI responses to 14- and 19-week fetal colon tissue, whereas leukocytes from control subjects did not. Neither group responded positively to 21-week fetal organs. Criss-cross experiments showed that the fetal antigen was organ specific. Multiparous pregnant women showed positive LAI responses to cancer extracts but not to extracts from normal tissues of the same organ. The pattern of the LAI response was bell-shaped. Positive LAI responses to lung and breast cancer were detected at 4 to 7 months gestation and peaked at 5 months. To the fetal colon, LAI positive responses were detected at 5 to 8 months gestation, with the peak response at 6 months. The results indicate that OSN of cancers are also expressed by fetal organs and sufficient antigen is shed by fetal organs to sensitize pregnant women. Older fetal organs (21 weeks) and adult organs do not express an immunogenic or antigenic OSN.Supported by a grant from the National Cancer Institute of Canada     

Lack of leukocyte migration inhibition by hepatitis B surface antigen in hepatocellular carcinoma patients.

Soluble part of hepatocellular carcinoma (HCC) tissue extracts with or without hepatitis B surface antigen (HBsAg) was tested against leukocytes of 13 histologically confirmed HCC patients. Inhibition of leukocyte migration was observed in 9 out of 13 cases when tested by soluble HCC extract containing HBsAg, while inhibition of lukocyte migration was observed in 8 out of 13 cases when tested by solublp greater than 0.05, by Fisher's exact test). In the meantime, soluble HCC extract with or without HBsAg did not significantly cause inhibition of leukocyte migration in 12 non-HCC patients. Therefore, it is concluded that inhibition of leukocyte migration in HCC patients is caused by the tumor-associated antigen, not caused by HBsAg.     

Leukocyte dependence of platelet adhesion in postcapillary venules

Reperfusion of ischemic tissues results in development of a proinflammatory, prothrombogenic phenotype, culminating in the recruitment of leukocytes and platelets within postcapillary venules. Recent studies have indicated an interdependence of platelet and leukocyte adhesion, suggesting that heterotypic blood cell interactions may account for postischemic platelet recruitment. The objectives of this study were to 1) determine whether ischemia-reperfusion (I/R)-induced platelet recruitment is leukocyte dependent and 2) quantify the contributions of leukocytes and endothelial cells in this platelet recruitment. Intravital microscopy was used to monitor the recruitment of fluorescently labeled platelets in postcapillary venules of the small intestine after 45-min ischemia and 4-h reperfusion. To assess the leukocyte dependence of platelet adhesion, platelets from wild-type mice were infused into mice deficient in neutrophils and/or lymphocytes and mice deficient in key leukocyte adhesion molecules (CD18 and ICAM-1). These antileukocyte strategies resulted in significantly reduced platelet recruitment. Simultaneous visualization of platelets and leukocytes enabled quantification of leukocyte-dependent and endothelium-dependent platelet adhesion. It was observed that in wild-type animals 74% of I/R-induced platelet adhesion was a result of platelet-leukocyte interactions. Although the majority of adherent platelets were associated with leukocytes, <50% of adherent leukocytes were platelet bearing, suggesting that not all adherent leukocytes support platelet adhesion. These results are consistent with leukocytes playing a major role in supporting I/R-induced platelet adhesion.     

Electron microscopy for the diagnosis of tumours.

The role of leukocyte transfusions in the prevention and treatment of infections in adults with granulocytopenia was investigated. Leukocytes were obtained from healthy volunteers by continuous-flow centrifugation. Histocompatibility antigen (HLA)-matched leukocytes were used to assess the prophylactic value of leukocyte transfusions. Seven patients with acute myelogenous leukemia received HLA-matched leukocytes during the period of maximal granulocytopenia associated with initial remission induction therapy; 20 concurrently treated patients who did not receive leukocyte transfusions were the control group. The patients receiving HLA-matched leukocytes had significantly fewer (P = 0.043) infectious episodes (not bacteriologically proven) during the study period, and remission occurred in 5 of the 7, compared with 10 of the 20 controls. In addition, 52 series of two or more ABO-compatible transfusions were given to 50 patients with proven infection or elevated temperature presumed due to infection and a granulocyte count of less than 0.5 X 10(9)/L. Response, indicated by a decrease in temperature, occurred in 23 patients. Leukocyte transfusions thus have an important adjuvant role in the management of patients with severe granulocytopenia.     

The human leukocyte-adhesion ligand, intercellular-adhesion molecule 2. Expression and characterization of the protein

The leukocyte cell-adhesion receptors, complexes of the cluster of differentiation antigen 11a with cluster of differentiation antigen 18 (CD11a/CD18), cluster of differentiation antigen 11b with cluster of differentiation antigen 18 (CD11b CD18) and cluster of differentiation antigen 11c with cluster of differentiation antigen 18 (CD11c CD18), are of major importance in several leukocyte functions. Previously a cellular ligand named intercellular-adhesion molecule 1 (ICAM-1) was identified, isolated and extensively characterized. Recently a second similar molecule, intercellular-adhesion molecule 2 (ICAM-2), was found by a functional DNA-cloning method. We have now synthesized the ICAM-2 DNA by the polymerase chain reaction (PCR), sequenced it, and transferred it into mammalian and bacterial expression vectors. A functional leukocyte-binding glycoprotein was obtained by transfection of COS-1 cells. A soluble protein-A - ICAM-2 fusion protein was made in Escherichia coli, purified and used for antiserum production. The antiserum precipitated a cell-surface protein with an apparent molecular mass of 55 kDa from ICAM-2 transfected COS-1 cells, leukocytes and endothelial cells, and inhibited leukocyte binding to transfected COS-1 cells. The bacterial fusion protein, lacking carbohydrate, specifically bound to leukocyte receptors.     

Paradoxical attenuation of leukocyte rolling in response to ischemia- reperfusion and extracorporeal blood circulation in inflamed tissue

In contrast to acute preparations such as the exteriorized mesentery or the cremaster muscle, chronically instrumented chamber models allow one to study the microcirculation under "physiological" conditions, i.e., in the absence of trauma-induced leukocyte rolling along the venular endothelium. To underscore the importance of studying the naive microcirculation, we implanted titanium dorsal skinfold chambers in hamsters and used intravital fluorescence microscopy to study venular leukocyte rolling in response to ischemia-reperfusion injury or extracorporeal blood circulation. The experiments were performed in chambers that fulfilled all well-established criteria for a physiological microcirculation as well as in chambers that showed various extents of leukocyte rolling due to trauma, hemorrhage, or inflammation. In ideal chambers with a physiological microcirculation (<30 rolling leukocytes/mm vessel circumference in 30 s), ischemia-reperfusion injury and extracorporeal blood circulation significantly stimulated leukocyte rolling along the venular endothelium and, subsequently, firm leukocyte adhesion. In contrast, both stimuli failed to elicit leukocyte rolling in borderline chambers (30-100 leukocytes/mm), and in blatantly inflamed chambers with yet higher numbers of rolling leukocytes at baseline (>100 leukocytes/mm), we observed a paradoxical reduction of leukocyte rolling after ischemia-reperfusion injury or extracorporeal blood circulation. A similar effect was observed when we superfused leukotriene B4 (LTB4) onto the chamber tissue. The initial increase in leukocyte rolling in response to an LTB4 challenge was reversed by a second superfusion 90 min later. These observations underscore 1) the benefit of studying leukocyte-endothelial cell interaction in chronically instrumented chamber models and 2) the necessity to strictly adhere to well-established criteria of a physiological microcirculation.