Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (Fc epsilon RI).

It has been suggested that epidermal Langerhans cells (LC) bearing immunoglobulin E (IgE) may be involved in the genesis of atopic disease. The identity of the IgE receptor(s) on LC remained unclear, although it represents a crucial point in understanding cellular events linked to the binding of allergens to LC via IgE. In this report, we demonstrate that epidermal LC express the high affinity receptor for the Fc fragment of IgE (Fc epsilon RI) which has, so far, only been described on mast cells and basophils. Epidermal LC react with antibodies specific for the alpha subunit of the tetrameric (alpha, beta, 2 gamma) Fc epsilon RI. Specific transcripts for Fc epsilon RI alpha and Fc epsilon RI gamma were detected in LC and correspond to those of human basophils and of the human basophil cell line KU812. Furthermore, human basophils, KU812 cells, and LC express the putative beta subunit. Thus human LC express the complete structure of Fc epsilon RI. This finding opens new perspectives in the putative functional role of this structure on antigen-presenting cells.

Release of soluble Fc gamma RII/CD32 molecules by human Langerhans cells: a subtle balance between shedding and secretion?

Freshly isolated human Langerhans cells (LC) express two forms of Fc gamma RII: a membrane-associated form detected by monoclonal antibody (MoAb) anti-CD32, which recognize an extracytoplasmic epitope of the molecule, and a soluble secreted form, whose existence is suggested by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments. Indeed, RT-PCR performed on total LC RNA reveals the presence of two Fc gamma RIIA mRNA, one encoding the FC gamma RIIA with a transmembrane region (membranous form) and the other without this region (soluble form). Densitometry studies performed on the two PCR products reveal that the ratio between the membranous form and the soluble secreted form is about 1.5. LC maintained in culture for 24-48 h lose the major part of their membrane Fc gamma RII expression (shown by flow cytometry) and release soluble Fc gamma RII molecules (revealed by dot-blot assay), but maintain the same ratio of the two Fc gamma RIIA mRNA. The disappearance of the membrane-associated Fc gamma RII may be explained either by modification of its recycling pathway or by proteolytic cleavage of the receptor at the cell surface. Thus, soluble Fc gamma RII molecules generated during LC culture may result from proteolytic cleavage of the cell-surface receptor and/or secretion of a soluble form derived from the translation of an alternate spliced mRNA. Interestingly, addition of TNF-alpha (10 ng/ml) to the culture medium i) maintains the expression of the membranous form, which can be detected on the LC surface at the same level as on freshly isolated LC, and ii) reverses the ratio (to 0.6) of the two Fc gamma RII mRNA, the mRNA encoding the soluble form becoming predominant. Thus, TNF-alpha seems to modify the expression of the Fc gamma RII at the mRNA level, favoring the secretion of soluble Fc gamma RII molecules, and changes the fate of the membranous Fc gamma RII.

Role of the leucine-rich domain of platelet GPIbalpha in correct post-translational processing--the Nancy I Bernard-Soulier mutation expressed on CHO cells.

The mechanisms governing the biosynthesis and surface expression of platelet adhesive receptors on parent megakaryocytes are as yet poorly understood. In particular, the assembly and processing of the multisubunit glycoprotein (GP) Ib-IX-V complex, a receptor for von Willebrand factor (vWf) is not fully understood. In the present work, these questions were addressed by reproducing a natural mutation of GPIbalpha found in a variant case of Bernard-Soulier syndrome (Nancy I), due to the deletion of leucine 179 in the seventh leucine-rich repeat of the polypeptide. Wild type and mutated GPIbalpha were transfected into CHO cells expressing GPlbbeta and GPIX. Flow cytometry showed surface expression of the three subunits of both GPIb-IX complexes, but GPlbalphadeltaLeu was present at lower levels (20-40%) and was recognized only by a sub class of monoclonal antibodies which epitopes were not modified by the mutation. These properties reproduce the defect found in the patient's platelets, demonstrating the causative nature of the mutation and validate the use of the CHO cells model. Biochemical studies were performed in an attempt to elucidate the mechanism of the conformational change of GPIbalphadeltaLeu. They unexpectedly revealed a major glycosylation deficiency of the mutated GPIbalpha leading to a 40% decrease in molecular weight. The other two subunits of the complex were however normal and present at the plasma membrane. The deletion led to complete functional deficiency with lack of vWf binding of CHOalphadeltaLeu transfected cells in the presence of botrocetin and defective adhesion to a vWf coated surface under static conditions. Finally, in contrast to normal CHOalphabetaIX cells, which displayed rolling and deceleration when perfused over a vWf surface, CHOalphadeltaLeubetaIX cells were unable to roll over or attach to a vWf substratum. These results show that the integrity of the leucine-rich region of GPIbalpha is essential for normal processing and function of the GPIb-IX complex. In addition, these results obtained in a cellular system supported the suspected role of the macroglycopeptide region of GPIbalpha in maintaining a suitable conformation of this multisubunit receptor to perform its adhesive function.

A novel monoclonal antibody against the extracellular domain of GPIbbeta modulates vWF mediated platelet adhesion.

GPIbbeta is disulfide-linked to GPIbalpha to form GPIb, a platelet receptor for von Willebrand factor (vWF). GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex, the exact function of GPIbbeta is not well established due to a lack of suitable ligands or antibodies. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIbbeta subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM.1 bound to GPIbbeta deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbbeta, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbbeta in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbbeta.

Role of cyclic AMP in promoting the thromboresistance of human endothelial cells by enhancing thrombomodulin and decreasing tissue factor activities.

1. The effects of forskolin, prostaglandin E1 (PGE1), dibutyryl cyclic AMP (db cyclic AMP), dibutyryl cyclic GMP (db cyclic GMP) and 3-isobutyl-l-methyl-xanthine (IBMX) were investigated on the expression of tissue factor and thrombomodulin activities on the surface of human saphenous vein endothelial cells (HSVEC) in culture. 2. Forskolin (10(-6) to 10(-4) M), PGE1 (10(-7) to 10(-5) M) and db cyclic AMP (10(-4) to 10(-3) M) caused a concentration-dependent decrease of cytokine-induced tissue factor activity. 3. Similar concentrations of forskolin, PGE1 and db cyclic AMP enhanced significantly constitutive thrombomodulin activity and reversed the decrease of this activity caused by interleukin-1 (IL-1). 4. IBMX (10(-4) M) decreased tissue factor activity and enhanced the effect of forskolin on tissue factor and thrombomodulin activities. 5. Forskolin (10(-4) M) decreased the IL-1-induced tissue factor mRNA and increased the thrombomodulin mRNA level. IL-1 did not change the thrombomodulin mRNA level after 2 h of incubation with HSVEC in culture. 6. Dibutyryl cyclic GMP (10(-4) M to 10(-3) M) did not influence tissue factor or thrombomodulin activity. 7. Our data suggest that elevation of intracellular cyclic AMP levels may participate in the regulation of tissue factor and thrombomodulin expression, thus contributing to promote or restore antithrombotic properties of the endothelium.

Biochemical and molecular basis of Bernard-Soulier syndrome: a review.

Bernard-Soulier syndrome (BSS) is a rare hereditary recessive autosomal bleeding disorder characterized by a prolonged bleeding time, giant platelets, thrombocytopenia, normal platelet aggregation in response to ADP and no agglutination in response to ristocetin. This disease is due to absence or abnormality of the platelet membrane glycoprotein GPIb-IX-V, the receptor for von Willebrand factor. All four genes encoding the complex have been cloned and 17 forms of BSS have to date been characterized at the functional, immunological and molecular levels. The mutations can be divided into two main groups. Firstly, mutations located in leucine rich repeats (LRR), responsible for conformational modifications of the molecule, in some cases higher sensitivity to proteases and loss of adhesive function of the receptor, which is expressed at lower than normal levels at the platelet membrane. When mutations affect the LRR of GPIbalpha, the presence of the other chains varies from normal to residual amounts. When mutations affect the LRR of GPIX, expression of the other chains is strongly diminished, suggesting that GPIX plays a major role in the stability of the complex. A second type of mutations leads to synthesis of a truncated molecule lacking the transmembrane domain and absence of its expression at the platelet surface, while the other chains are present in residual amounts. Expression of recombinant proteins in eukaryotic cells has recently confirmed the results derived from studies of natural mutations. Separate expression of each chain can be obtained, although the presence of all subunits is required for full expression.

Ethnic polymorphism of DXS52 (St 14) locus linked to coagulation factor VIII gene in Argentina.

Sixty-five individuals belonging to 16 argentinian families of hemophilia A were studied using the St 14 probe (DXS52 locus). This probe is widely used for carrier detection and prenatal diagnosis, despite the risk of recombination between the factor VIII gene and the DXS52 locus, because of its high informativity. The families are divided in two groups: one group constituted only of metis of Indians according to interview and morphotype and a second group of caucasoids (Spanish essentially and Italian). In this study we have shown some ethnic variations of the TaqI RFLPs in the DXS52 locus. In the allelic system I, (which alleles are numbered from 1 to 8) we have noted an over representation of the larger alleles (2 and 3) and of the allele 8 in both Argentinian groups when compared to the caucasian population already studied in our laboratory. The additional polymorphic TaqI site giving the beta band in the system II (alpha and beta bands) is found more frequently in the Argentinian families than in Caucasians. Some other additional polymorphic sites have been found in generally constant bands giving additional allelic systems, in metis families.

The alpha(IIb)beta(3) integrin and GPIb-V-IX complex identify distinct stages in the maturation of CD34(+) cord blood cells to megakaryocytes.

Megakaryocytopoiesis is a complex multistep process involving cell division, endoreplication, and maturation and resulting in the release of platelets into the blood circulation. Megakaryocytes (MK) progressively express lineage-restricted proteins, some of which play essential roles in platelet physiology. Glycoprotein (GP)Ib-V-IX (CD42) and GPIIb (CD41) are examples of MK-specific proteins having receptor properties essential for platelet adhesion and aggregation. This study defined the progressive expression of the GPIb-V-IX complex during in vitro MK maturation and compared it to that of GPIIb, an early MK marker. Human cord blood CD34(+) progenitor cells were cultured in the presence of cytokines inducing megakaryocytic differentiation. GPIb-V-IX expression appeared at day 3 of culture and was strictly dependent on MK cytokine induction, whereas GPIIb was already present in immature CD34(+) cells. Analysis by flow cytometry and of the messenger RNA level both showed that GPV appeared 1 day later than GPIb-IX. Microscopy studies confirmed the late appearance of GPV, which was principally localized in the cytoplasm when GPIb-IX was found on the cell surface, suggesting a delayed program of GPV synthesis and trafficking. Cell sorting studies revealed that the CD41(+)GPV(+) population contained 4N and 8N cells at day 7, and was less effective than CD41(+)GPV(-) cells in generating burst-forming units of erythrocytes or MK colonies. This study shows that the subunits of the GPIb-V-IX complex represent unique surface markers of MK maturation. The genes coding for GPIb-IX and GPV are useful tools to study megakaryocytopoiesis and for tissue-specific or conditional expression in mature MK and platelets. (Blood. 2000;96:4169-4177)

Thrombin regulates tissue factor and thrombomodulin mRNA levels and activities in human saphenous vein endothelial cells by distinct mechanisms.

The effects of thrombin, D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK)-inhibited thrombin, and thrombin receptor agonist peptide, SFLLRNPNDKYEPF (SFLL, a portion of the receptor unmasked after thrombin cleavage), on the expression of tissue factor (TF) and thrombomodulin by human saphenous vein endothelial cells (HSVECs) in culture were studied. Unstimulated cells contained very low amounts of TF mRNA as measured by the reverse transcriptase-PCR method. Thrombin treatment increased TF mRNA to 8.0 +/- 1.9 (n = 3) times the control level. The increase was detectable within 2 h and declined to near basal level by 6 h. Induction of TF mRNA was not blocked by cycloheximide, treatment with cycloheximide alone also increased TF mRNA levels, and thrombin in combination with cycloheximide further enhanced the accumulation of TF mRNA. Thrombin caused a 14.5 +/- 1.5-fold (n = 5) increase in TF activity on the surface of HSVECs and a 20.5 +/- 1.4-fold (mean +/- S.D., n = 2) increase in the extracellular matrix. The thrombin-induced effects on TF synthesis could be fully reproduced by the thrombin receptor agonist peptide, SFLL, whereas PPACK-inhibited thrombin did not influence TF expression. Thrombin increased thrombomodulin mRNA to 190 +/- 39% (n = 5) of control levels, whereas PPACK-inhibited thrombin or SFLL did not influence thrombomodulin mRNA levels. In contrast, surface-bound thrombomodulin cofactor activity and thrombomodulin antigen in the cell lysates did not change over 24 h of incubation with thrombin. However, thrombin caused a 2-fold increase in thrombomodulin antigen released into the conditioned medium, and immunoelectron microscopy of HSVECs also demonstrated the presence of thrombomodulin vesicles close to the luminal cell surface in thrombin-treated cultures. The Western blot pattern thrombomodulin in the conditioned medium of untreated and thrombin-treated cells was found to be similar, and soluble thrombomodulin occurred mainly as fragments of the cell-associated form. We conclude that the transcriptional control by thrombin causes an increase in both TF and thrombomodulin mRNA. The increase in TF mRNA levels is also paralleled by an increase in surface expression, is dependent on the proteolytic activity of thrombin, and is mediated by the same receptor as the recently cloned thrombin receptor in platelets. Up-regulation of thrombomodulin mRNA levels by thrombin is distinct from this pathway and is associated with unchanged expression on the cell surface.

Functional characterization of the human platelet glycoprotein V gene promoter: A specific marker of late megakaryocytic differentiation.

Glycoprotein V (GPV), a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin, is specifically found in platelets and mature megakaryocytes. Studies of the GPV gene can therefore provide insight into the mechanisms governing megakaryocyte differentiation. The human GPV promoter was isolated, and elements important for its tissue specific transcriptional activity were localized using systematic DNase I protection and reporter deletion assays. A -1413/+25 fragment inserted into a luciferase reporter construct displayed promoter activity in Dami and HEL but not in K562, HL60, or HeLa cells. Progressive 5' to 3' deletion showed a putative enhancer region in the -1413/-903 segment that contained closely spaced GATA and Ets sites protected from DNase I digestion in Dami extracts. Regions similar to a GPIIb gene repressor were found at -816 and -610, with the first exhibiting repressor activity in Dami and HEL cells and the second protected from DNAse I. Deletions from -362 to -103, an area containing protected sites for Sp1, STAT, and GATA, induced a progressive decrease in activity. The -103/+1 fragment, bearing a proximal Ets footprinted site and a GATA/Ets tandem footprint, displayed 75% activity relative to the full-length promoter and retained cell specificity. In summary, this work defines several regions of the GPV gene promoter important for its activity. It contains megakaryocyte-specific signals, including erythro-megakaryocytic GATA, and Ets cis-acting elements, GPIIb-like repressor domains, and binding sites for ubiquitous factors such as Sp1, ETF, and STAT.

Common intragenic and extragenic polymorphisms of blood coagulation factors VIII and IX are different in Chinese and Caucasian populations.

In order to examine the possibilities of carrier detection and prenatal diagnosis in hemophilia A and B in the Chinese region of Suzhou, we analyzed four different RFLPs within the factor IX gene and two intragenic RFLPs and one extragenic RFLP for the factor VIII gene. The results obtained show important differences between the Chinese and Caucasian populations. No polymorphism was found within the factor IX gene in the Chinese population and the informativity obtained for the factor VIII gene was quite different between the two populations for each RFLP studied.

Angiogenesis inhibitor SR 25989 upregulates thrombospondin-1 expression in human vascular endothelial cells and foreskin fibroblasts.

The aim of this work was to evaluate the effects of SR 25989, a member of the thienopyridine family devoid of antiplatelet activity but possessing anti-angiogenic properties, on the regulation of proteins involved in matrix remodeling during wound healing or tumor progression. Human endothelial cells grown in the presence of SR 25989 showed moderate increases in the production of activators (tissue plasminogen activator and urokinase) and one inhibitor (plasminogen activator inhibitor type 1) of fibrinolysis, together with a significant rise in intracellular thrombospondin-1. SR 25989 induced a similar increase in thrombospondin-1 in human foreskin fibroblasts. This over-expression of thrombospondin-1 was correlated to a decrease in cell density. A concomitant increase in the tumor suppressor gene protein p53 was observed in endothelial cells and in fibroblasts, in which the slowing down of proliferation could be related to an accumulation of cells in the S and G2/M phases of the cell cycle. Northern blot analysis revealed a temporary rise in thrombospondin-1 transcripts, followed by a decrease along with a moderate increase in p53 transcripts. Thus the anti-angiogenic properties of SR 25989 appear to result from an upregulation of thrombospondin-1 which is possibly mediated by p53. The thienopyridine SR 25989 could therefore be a good candidate for adjuvant anti-angiogenic therapy in cancer.

Release of Fc gamma RIIa2 by activated platelets and inhibition of anti-CD9-mediated platelet aggregation by recombinant Fc gamma RIIa2.

Thrombin-activated human platelets and megakaryocyte cell lines release soluble Fc gamma RII (Fc gamma RIIa2) containing the extracellular and intracellular regions of Fc gamma RIIa1, but lacking the transmembrane domain. Use of polyclonal antibodies directed either against the entire intracytoplasmic tail, or against a peptide located near the C-terminal part of the intracellular region of Fc gamma RIIa2, showed the presence of both a complete form of Fc gamma RIIa2 and a C-terminal truncated form in supernatants of platelets after release of their alpha granule contents and in culture supernatants of megakaryocyte cell lines. Furthermore, recombinant Fc gamma RIIa2 inhibited in a dose-dependent manner Fc-dependent anti-CD9 antibody-induced platelet aggregation. Thus, release of Fc gamma RIIa2 by activated platelets could play an important role in the regulation of platelet activation by immune complexes.

A three-base deletion removing a leucine residue in a leucine-rich repeat of platelet glycoprotein Ib alpha associated with a variant of Bernard-Soulier syndrome (Nancy I).

Leucine-rich repeats are conserved structural motifs present in the four components of the human platelet glycoprotein Ib/IX/V complex receptor for the adhesive protein von Willebrand factor. The absence or abnormality of this complex is responsible for Bernard-Soulier disease, an autosomal recessive bleeding disorder. We report a deletion of leucine 179, located in a highly conserved position of the seventh leucine-rich repeat of GPIb alpha, found in a variant form of Bernard-Soulier disease (Bernard-Soulier Nancy I). Three affected siblings of a family were characterized by absence of ristocetin-induced platelet agglutination, although ADP aggregation was normal. Flow cytometry studies showed detectable amounts of all four members of the GPIb/IX/V complex on the surface of the patients' platelets. Western blotting revealed normal levels of GPIX, decreased levels of GPIb beta and GPV, and < 1% of GPIb alpha. RT-PCR studies showed the presence of mRNA coding for GPIb alpha, GPIb beta, GPIX and GPV. Sequencing showed a three-base deletion which results in the absence of a leucine residue, highly conserved across the seven leucine-rich repeats of GPIb alpha and also within the other members of the leucine-rich glycoprotein family. The absence of the leucine 179 in a patient's GPIb alpha is believed to cause a conformational change in the protein which would account for the lack of binding of most of the MoAbs tested and would be responsible for the absence of von Willebrand factor binding. These results point to the leucine-rich region of GPIb alpha as being required for the correct exposure of the von Willebrand binding site as well as for the correct assembly and stability of the GPIb/IX/V complex on the platelet surface.

DNA amplification of HIV genome in hemophiliacs and in newborns from seropositive mothers.

We evaluated the validity of DNA enzymatic amplification (PCR) in a population at risk for HIV-1 infection, consisting of hemophiliacs and children born to seropositive mothers. All but one of the seropositive hemophiliacs and controls were found positive with the three sets of primers. All the seronegative patients and controls were found negative in PCR. No correlation with the anti-nef serology was found, one seropositive being anti-nef negative and three seronegative anti-nef positive. The results obtained with PCR are in good agreement with classical serology, and this would suggest that the possible period of latency may not be as long as suspected. No seroconversion has been described in hemophiliacs since solvent-detergent inactivated blood products have been in use, and seronegative hemophiliacs no longer constitute a population at risk. Studies on seronegative sexual partners of seropositive patients would be of great interest. For newborns from seropositive mothers, PCR is the only possible technique in early age before seronegativation of the healthy children. Further studies will be required to determine the fiability and sensitivity of the test.

Identification, in mouse macrophages and in serum, of a soluble receptor for the Fc portion of IgG (Fc gamma R) encoded by an alternatively spliced transcript of the Fc gamma RII gene.

Low affinity Fc gamma R are a heterogeneous group of glycoproteins which exist in transmembrane (TM) as well as in soluble forms. Two membrane isoforms of the murine type II Fc gamma R, Fc gamma RIIb1 and Fc gamma RIIb2, have been described. They result from the translation of alternatively spliced pre-mRNA, Fc gamma RIIb2 lacking sequences of the first intracytoplasmic domain (IC1). Soluble forms of Fc gamma R (sFc gamma R) have previously been shown to result from proteolysis of membrane receptors. We report here the identification, in macrophages, of a mRNA derived from the Fc gamma RII gene by splicing exons encoding the TM and IC1 domains, i.e. corresponding to a TM-deleted Fc gamma RIIb2 mRNA. A soluble protein possibly encoded by this mRNA was identified in macrophage supernatants. In accordance with Fc gamma R nomenclature, we propose to name this new Fc gamma RII isoform Fc gamma RIIb3. It is the most abundant sFc gamma R present in serum, as compared with sFc gamma R resulting from cleavage of membrane Fc gamma R.