Human keratinocytes release the endogenous beta-galactoside-binding soluble lectin immunoglobulin E (IgE-binding protein) which binds to Langerhans cells where it modulates their binding capacity for IgE glycoforms.

A better understanding of the pathophysiological role of Langerhans cells (LC) in atopic diseases is dictated by the characterization of the structures involved in immunoglobulin (IgE)-binding on their cell surface. We previously reported that human LC express the high affinity receptor for IgE (Fc epsilon RI), as well as the low affinity receptor for IgE (Fc epsilon RII/CD23). In the present study, we document the presence of a third IgE-binding structure on human LC, the IgE-binding protein (epsilon BP), an endogenous soluble beta-galactoside binding lectin. Immunohistochemical studies performed on normal human skin revealed an anti-epsilon BP reactivity in the cytoplasm of keratinocytes and in that of acinous cells of eccrine sweat glands. epsilon BP was also found on the cell surface of LC, as shown by anti-epsilon BP/anti-CD1a double labeling and flow cytometric analysis. Anti-epsilon BP binding to the surface of LC was completely abolished by preincubation with lactose and restored by addition of recombinant human epsilon BP, indicating that epsilon BP binds to LC surface by virtue of its lectin property. Immunoblot analysis of anti-epsilon BP-reactive material in keratinocytes and purified LC disclosed a protein with an apparent molecular weight of 33,000 consistent with epsilon BP. Interestingly, mRNA transcripts for epsilon BP were detected only in keratinocytes but not in purified LC isolated from normal skin. epsilon BP was found to be released in culture supernatants of keratinocytes. Incubation of LC with these supernatants resulted in epsilon BP-binding to LC surface via protein-carbohydrate interaction. Most importantly, we could show that binding of human myeloma IgE to LC was inhibited by epsilon BP. In contrast, neuraminidase-treated human myeloma IgE binds to LC only in the presence of epsilon BP. In situ binding studies revealed that keratinocytes, although containing epsilon BP intracytoplasmatically, failed to exhibit any IgE-binding properties. Collectively, our results suggest that human keratinocytes produce the beta-galactoside-binding lectin epsilon BP, which subsequently binds to the surface of LC where it is functional in modulating their binding capacity for IgE glycoforms.

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.

An invariant T cell receptor alpha chain defines a novel TAP-independent major histocompatibility complex class Ib-restricted alpha/beta T cell subpopulation in mammals.

We describe here a new subset of T cells, found in humans, mice, and cattle. These cells bear a canonical T cell receptor (TCR) alpha chain containing hAV7S2 and AJ33 in humans and the homologous AV19-AJ33 in mice and cattle with a CDR3 of constant length. These T cells are CD4(-)CD8(-) double-negative (DN) T cells in the three species and also CD8alphaalpha in humans. In humans, their frequency was approximately 1/10 in DN, 1/50 in CD8alpha+, and 1/6,000 in CD4(+) lymphocytes, and they display an activated/memory phenotype (CD45RAloCD45RO+). They preferentially use hBV2S1 and hBV13 segments and have an oligoclonal Vbeta repertoire suggesting peripheral expansions. These cells were present in major histocompatibility complex (MHC) class II- and transporter associated with antigen processing (TAP)-deficient humans and mice and also in classical MHC class I- and CD1-deficient mice but were absent from beta2-microglobulin-deficient mice, indicating their probable selection by a nonclassical MHC class Ib molecule distinct from CD1. The conservation between mammalian species, the abundance, and the unique selection pattern suggest an important role for cells using this novel canonical TCR alpha chain.

Activity and phenotype of natural killer cells in peptide transporter (TAP)-deficient patients (type I bare lymphocyte syndrome).

In this paper we describe the function and phenotype of natural killer (NK) lymphocytes from HLA class I-deficient patients. These cells are, as has been previously reported, unable to lyse HLA class I- K562 cells, but are able to perform antibody-dependent cellular cytotoxicity (ADCC), although with lower efficiency as compared to NK cells from normal individuals. Transporter associated to antigen processing (TAP)- NK cells proliferate when cultured in the presence of lymphoblastoid B cells (B-LCs) and interleukin 2 and develop a spectrum of cytotoxicity similar to that of activated normal NK cells. Importantly, activation of the TAP- NK cells induces strong cytotoxicity to autologous B-LCs. Analysis of the phenotype of circulating TAP- NK lymphocytes showed them to display a normal diverse repertoire of HLA class I-specific NK receptors. These receptors were expressed at normal levels, apart from the CD94-NKG2A complex, which appeared to be overexpressed. This latter finding could reflect an adaptation to the low expression of HLA class I molecules. Finally, functional analyses indicated that the inhibitory receptors in TAP- individuals can transduce inhibitory signals. Our results suggest that in vivo, the NK cells of TAP- patients could participate in immune defense, at least through ADCC, but upon activation, may be involved in autoimmune processes.

Homozygous human TAP peptide transporter mutation in HLA class I deficiency.

Human lymphocyte antigen (HLA) class I proteins of the major histocompatibility complex are largely dependent for expression on small peptides supplied to them by transporter associated with antigen processing (TAP) protein. An inherited human deficiency in the TAP transporter was identified in two siblings suffering from recurrent respiratory bacterial infections. The expression on the cell surface of class I proteins was very low, whereas that of CD1a was normal, and the cytotoxicity of natural killer cells was affected. In addition, CD8+ alpha beta T cells were present in low but significant numbers and were cytotoxic in the most severely affected sibling, who also showed an increase in CD4+CD8+ T cells and gamma delta T cells.

Carrier detection of Hemophilia B by using a restriction site polymorphism associated with the coagulation Factor IX gene.

The cloned complementary DNA for coagulation Factor IX (FIX) detects a frequent restriction fragment length polymorphism (RFLP) in human genomic DNAs digested with the restriction endonuclease Taq I. This genetic marker was used, in parallel with coagulation and immunological assays, to follow the segregation of an abnormal FIX gene in a large Hemophilia B family. Among the six potential female carriers, functional assays showed that four had a high probability, and two a low probability of being carriers. Analysis at the DNA level with the cDNA probe was informative in five of the six cases, and in all these five the diagnosis of carrier state was definitively confirmed. This demonstrates the feasibility of using linkage analysis at the DNA level for the genetic screening of Hemophilia B. This method has the advantages over conventional assays of giving a diagnosis of certainty, and of being applicable to early prenatal diagnosis using biopsies of trophoblast villi. At present, the single known polymorphism associated with the FIX gene restricts the application of linkage analysis to informative cases (40%), but findings of additional RFLPs in this region should improve this figure.

HLA class I deficiencies due to mutations in subunit 1 of the peptide transporter TAP1.

The transporter associated with antigen processing (TAP), which is composed of two subunits (TAP1 and TAP2) that have different biochemical and functional properties, plays a key role in peptide loading and the cell surface expression of HLA class I molecules. Three cases of HLA class I deficiency have previously been shown to result from the absence of a functional TAP2 subunit. In the present study, we analyzed two cases displaying not only the typical lung syndrome of HLA class I deficiency but also skin lesions, and found these patients to be TAP1-deficient. This defect leads to unstable HLA class I molecules and their retention in the endoplasmic reticulum. However, the absence of TAP1 is compatible with life and does not seem to result in higher susceptibility to viral infections than TAP2 deficiency. This work also reveals that vasculitis is often observed in HLA class I-deficient patients.

Platelets are dispensable for antibody-mediated transfusion-related acute lung injury in the mouse.

UNLABELLED: Essentials Role of platelets in immunological transfusion-related acute lung injury (TRALI) is debated. Immunological TRALI was tested in mice exhibiting severe thrombocytopenia or platelet dysfunction. Platelets are required to prevent lung hemorrhage but not edema formation and respiratory distress. Platelets are dispensable for the initiation and development of TRALI. SUMMARY: Background Transfusion-related acute lung injury (TRALI) is a serious transfusion-related complication. Previous conflicting studies have indicated that platelets are either crucial or dispensable for TRALI. Objectives To evaluate the role of platelets in major histocompatibility complex (MHC) I-induced-TRALI. Methods Antibody-mediated TRALI was experimentally induced in mice by lipopolysaccharide priming followed by the administration of an anti-MHC I mAb. Results TRALI was tested in the context of severe thrombocytopenia provoked by the administration of diphtheria toxin (DT) in transgenic iDTR mice selectively expressing DT receptor in megakaryocytes. The pathologic responses occurring within the first 10 min following the injection of the anti-MHC I mAb, i.e. the severity of lung edema and the drop in aortic blood oxygenation, were similar in severely thrombocytopenic DT-iDTR and control mice. At later times, mortality was nevertheless increased in DT-iDTR mice, owing to lung hemorrhages. When less severe thrombocytopenia was induced with an antiplatelet mAb, TRALI started and developed similarly as in control mice, but hemorrhages were absent. Furthermore, when platelet functions were defective because of administration of aspirin or clopidogrel, or because of glycoprotein (GP)IIbIIIa deficiency, TRALI still developed but no lung hemorrhages were observed. In contrast, when GPVI was immunodepleted, TRALI still occurred, but was occasionally accompanied by hemorrhages. Conclusions Platelets are dispensable for the initiation and development of MHC I-induced TRALI. Although they do not protect against the disruption of the vascular endothelial cell barrier and the subsequent plasma leakage and edema formation, platelets are essential to prevent more serious damage resulting in hemorrhages in alveoli.

Clinical and immunological aspects of HLA class I deficiency.

Human leukocyte antigen (HLA) class I deficiency is a rare disease with remarkable clinical and biological heterogeneity. The spectrum of possible manifestations extends from the complete absence of symptoms to life-threatening disease conditions. It is usually diagnosed when HLA class I serological typing is unsuccessful; flow cytometric studies then reveal a severe reduction in the cell surface expression of HLA class I molecules (90-99% reduction compared to normal cells). In most cases to date, this low expression is due to a homozygous inactivating mutation in one of the two subunits of the transporter associated with antigen processing (TAP), critically involved in the peptide loading of HLA class I molecules. Although asymptomatic cases have been described, TAP deficiencies are usually characterized by chronic bacterial infections of the upper and lower airways, evolving to bronchiectasis, and in half of the cases, also skin ulcers with features of a chronic granulomatous inflammation. Despite the defect in HLA class-I-mediated presentation of viral antigens to cytotoxic T cells, the patients do not suffer from severe viral infections, presumably because of other efficient antiviral defence mechanisms such as antibodies, non-HLA-class-I-restricted cytotoxic effector cells and CD8+ T-cell responses to TAP-independent antigens. Treatment is at present exclusively symptomatic, and should particularly focus on the prevention of bronchiectasis, which requires early detection.

CD1a molecules traffic through the early recycling endosomal pathway in human Langerhans cells.

In this work, we studied the localization and traffic of CD1a molecules in human epidermal Langerhans cells and the ability of these cells to stimulate CD1a-restricted T cell clones. We found that CD1a was spontaneously internalized into freshly isolated Langerhans cells, where it was rapidly distributed to the early/sorting endosomes and then to the early/recycling endosomes. In the latter compartments, CD1a colocalized with Rab11, a small GTPase known to be involved in the recycling of transmembrane proteins from early endosomes to the cell surface. In the steady state, intracellular CD1a was mainly located in Rab11+ recycling endosomal compartments. When endocytosis was blocked, intracellular CD1a moved rapidly from the early/recycling endosomes to the cell surface where it accumulated. The resultant increase in the cell surface expression of CD1a enhanced the capacity of Langerhans cells to stimulate a CD1a-restricted T cell clone. These findings are consistent with a dynamic exchange of CD1a between recycling compartments and the plasma membrane and suggest that the antigen-presenting function of CD1a depends on its traffic through the early/recycling endosomal pathway.

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.

Use of synthetic oligonucleotides in gene isolation and manipulation.

Great progress has occurred in the techniques of synthesis of DNA molecules of defined sequences in terms of speed, length of the obtained oligonucleotides, and automation of the processes. Corresponding progress also occurred in the ways of using synthetic DNA in molecular biology and recombinant DNA research. Screening of cloned DNA sequence banks with long, unique oligonucleotides, provided a new approach to isolate the genes for proteins which are present in very small quantity. This technique can present considerable advantages over the more classical use of mixtures of oligonucleotides, in reducing the number of potentially positive clones on a primary screen, and enabling cloning with a minimum of amino acid sequence data. Synthetic oligonucleotides also provide the basis of a set of techniques for site-directed mutagenesis of DNA sequences. This allows the possibility of engineering the structure of particular proteins, and the properties of new variants can be tested by expressing the protein in a heterologous host. An example of this approach is the production of variants of human alpha 1-antitrypsin. A variant where valine replaces the methionine at the active site is equally active as an antielastase, but no longer susceptible to oxidative inactivation. A second variant, where arginine replaces the methionine, now functions as an antithrombin, but no longer inhibits elastase. Total gene synthesis is now feasible for larger and larger genes, and some of the recent strategies of whole gene synthesis are presented.

Detection and quantification of secreted soluble Fc gamma RIIA in human sera by an enzyme-linked immunosorbent assay.

Fc gamma RIIA can be produced in a soluble form that contains both the extracellular and intracellular regions of the receptor, due to an alternative splicing of the transmembrane domain-coding exon. We have developed an enzyme-linked immunosorbent assay (ELISA) that permits the specific detection and quantification in human sera of this secreted soluble Fc gamma RIIA. It uses the monoclonal antibody (MAb) IV.3 as capture antibody and rabbit polyclonal IgG directed against the intracellular region of Fc gamma RIIA as detector antibodies. The enzymatic reaction was amplified using an NADH/NAD+ amplification system. As little as 0.8-1.5 ng/ml (20-38 pM) of purified recombinant secreted Fc gamma RIIA could be detected. The serum levels of secreted sFc gamma RIIA ranged from 0 to 30 ng/ml in sera from 51 healthy donors. The mean value was 11.9 ng/ml +/- 6.55 (297 pM +/- 163) and the median value was 10.6 ng/ml (265 pM) (range: 0-764 pM).

In vitro carboxylation of a blood coagulation factor IX precursor produced by recombinant-DNA technology.

Blood coagulation factor IX (Christmas factor) is a plasma protein which is required for normal haemostasis. A functional deficiency of factor IX results in haemophilia B, a bleeding disorder which is generally treated by infusions of factor IX concentrates prepared from pooled human plasma. The use of human blood products is connected with the risk of transmitting viral agents responsible for diseases such as hepatitis B and AIDS. Recombinant DNA techniques may provide the means to produce the required proteins without exposing the patients to these risks and at lower costs. One of the problems which has to be overcome before recombinant factor IX can be used for therapeutical purposes is related to the vitamin K-dependent carboxylation of its 12 NH2-terminal glutamate residues. In cell cultures this carboxylation, which is required to render the protein its procoagulant activity, is far from complete, especially at high expression levels. In this paper we describe the in vitro carboxylation of non and/or partly carboxylated recombinant factor IX produced by transformed Chinese hamster ovary cells. The identity of the newly formed Gla residues was verified and it could be demonstrated that all carboxyl groups had been incorporated into the recombinant factor IX.

Human factor B. Complete cDNA sequence of the BF*S allele.

The gene of human complement factor B (BF) is located within the class III region of the major histocompatibility complex. The knowledge of the coding sequence of the BF gene rests on a set of partial sequence studies reported by various sources, and full-length sequences ascribed to specific alleles of this polymorphic complement component have not yet been published. Now, we have isolated and sequenced a collection of cDNA clones derived from BF*S, the major BF allele. We present an uninterrupted, allele-specific sequence of the entire coding region and the 3' untranslated segment of the cDNA. Extensive comparison of this and previously available sequence data was carried out, and a number of base substitutions were observed in relation to some of the earlier sequences. The possibility that these differences arise from polymorphism in the BF gene is discussed.

CD1 expression is not affected by human peptide transporter deficiency.

Conventional major histocompatibility complex class I molecules are highly polymorphic and present peptides to cytotoxic T cells. These peptides derive from the proteolytic degradation of endogenous proteins in the cytosol and are translocated into the endoplasmic reticulum by a peptide transporter consisting of two transporter associated with antigen processing (TAP) molecules. Absence of this transporter leads to the synthesis of unstable peptide free class I molecules that are weakly expressed on the cell surface. Mouse nonconventional class I molecules (class Ib) may also present TAP-dependent peptides. In humans, CD1 antigens are nonconventional class I molecules. Recently, we characterized a human HLA class I deficiency resulting from a homozygous TAP deficiency. We show here that CD1a and -c are normally expressed on epidermal Langerhans cells of the TAP-deficient patients, as are CD1a, -b, and -c on dendritic cells differentiated in vitro from monocytes. Moreover, the CD1a antigens present on the surface of the dendritic cells are functional, since they internalize by receptor-mediated endocytosis gold-labeled F(ab')2 fragments of an anti-CD1a mAb. This suggests either that CD1 molecules are empty molecules, that they are more stable than empty conventional class I proteins, or that CD1 molecules present TAP-independent peptides.

Functions of Fc receptors on human dendritic Langerhans cells.

Immature dendritic cells are antigen presenting cells highly specialized for capturing and processing foreign protein antigens. These cells express Fc gamma RII and Fc epsilon RI which, by their ability to internalize and use the endocytic pathway, increase their capacity to process antigens. Immature dendritic cells, such as epidermal Langerhans cells, also release soluble forms of Fc gamma RII. These latter molecules are likely to compete with the membrane-associated Fc gamma R to diminish or abrogate the capacity of dendritic cells to present immune complexes, as suggested by our in vitro experiments using both human and mouse epidermal Langerhans cells. However, when dendritic cells mature in vitro and become efficient stimulators of resting T cells, they rapidly down-regulate and sometimes completely abolish the expression of their membrane-associated Fc gamma R and Fc epsilon RI. Consequently, they lose or at least strongly diminish their capacity to capture immune complexes. At this stage, the release of soluble Fc gamma R by dendritic cells is also markedly diminished. One can hypothesize that the membrane-associated Fc gamma RII and the soluble Fc gamma RII are molecules expressed when dendritic cells are potent capturing and processing cells, the soluble Fc gamma RII molecule acting by competition as a negative regulatory element on the Fc gamma RII-mediated internalization of IgG-containing immune complexes. Thus, the expression of membrane-associated Fc gamma R and Fc epsilon RI, as well as the release of soluble Fc gamma R, would seem to characterize the immature stage of dendritic cells.

New insights in the structure and biology of the high affinity receptor for IgE (Fc epsilon RI) on human epidermal Langerhans cells.

The recent structural and functional analysis of the high affinity receptor for IgE (Fc epsilon RI) expressed on human epidermal Langerhans cells (LC) revealed new aspects of the biology of this structure. In contrast to basophils and mast cells where this receptor seems to be expressed constitutively at a constant level, the expression of Fc epsilon RI on LC varies on the donor and the inflammatory environment of the cells and lacks the classical beta-chain. This also implies functional differences most probably related to the expression level. Although the signalling pathway seems to be similar to that of basophils or mast cells, LC from individuals with atopic dermatitis are fully activated by receptor ligation while LC from normal individuals fail to exhibit calcium mobilization under the same conditions. Finally, LC from normal and atopic individuals use Fc epsilon RI to maximize antigen uptake via specific IgE and subsequent presentation to T cells. Thus, Fc epsilon RI expressed on LC differs in terms of structure and function from that expressed on effector cells of anaphylaxis.

Synthesis and regulation of complement components by human monocytes/macrophages and by acute monocytic leukemia.

Proteins of the complement system (C2, C3) are synthesized by human monocytes and macrophages, thus providing an important local source of these proteins in vivo which serve as a first-line host defense mechanism. In this study, we investigated the production of complement components C2, C4, and C9 by human monocytes/macrophages and by the pathologic cells of acute monocytic leukemia which represent a source of immature monocytic precursors. Human blood monocytes were collected and purified by cytapheresis and elutriation and leukemic cells by Ficoll gradient. Secretion of complement components was measured by a hemolytic assay. The evaluation of the mRNAs of the various complement components in the cells was performed by polymerase chain reaction (PCR) by adding 32P labeled deoxycytidinetriphosphate (dCTP) to the amplification step. Functional C2 was found to increase during in vitro maturation of macrophages up to the fourth week of culture. C2 mRNA was detected after amplification and increased during the maturation. Interferon-gamma (IFN-gamma) mediated a marked increase of the C2 mRNA. We found a decrease in synthesis of C4 mRNA during in vitro differentiation of human monocytes. The effect of IFN-gamma resulted in an increase in C4 mRNA. C9 mRNA was not detected although it was detected in the HepG2 hepatoma-derived cell line. Functional C2 was not detected by leukemic cells after 24 h of culture but little functional C4 was present in the cell supernatants. As they were by human monocytes and macrophages, C2 and C4 mRNAs were detected after amplification but C9 mRNAs were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble Fcgamma receptor, Fc gammaRIIa2, is present in two forms in human serum and is increased in patients: with stage C chronic lymphocytic leukemia.

Human Fcgamma receptor type II (FcgammaRII/CD32) can be produced in a soluble form, termed FcgammaRIIa2, which contains the extra- and intracellular regions of the receptor, due to an alternative splicing of the transmembrane domain-encoding exon. We show that human sera contain two forms of FcgammaRIIa2. A full-length secreted protein, which has a 32 kDa backbone, can be detected only in some sera while all sera contain a C-terminal truncated form, lacking part of the intracytoplasmic tail, and exhibiting a 24 kDa protein backbone. The 24 kDa form is significantly increased in sera from stage C patients with B chronic lymphocytic leukemia, compared to healthy donors, stage A and B CLL patients, or CLL patients in complete remission.