Mechanism of Platelet Activation and Hypercoagulability by Antithymocyte Globulins (ATG).

T cell depletion with antithymocyte globulins (ATG) can be complicated by thrombopenia and hypercoagulability. The underlying mechanism is still unclear. We found that binding of ATG to platelets caused platelet aggregation, α-granule release, membrane phosphatidylserine exposure and the rapid release of procoagulant platelet microvesicles (MV). Platelet activation and MV release were complement-dependent and required membrane insertion of C5b-8 but not stable lytic pore formation by C5b-9. ATG also activated platelets via binding to the low-affinity Fc gamma receptor FcγRII. However, only complement inhibition but not blockade of FcγRII prevented MV release and subsequent thrombin activation in plasma. In 19 hematopoietic stem cell and kidney transplant patients, ATG treatment resulted in thrombopenia and increased plasma levels of d-dimer and thrombin-antithrombin complexes. Flow cytometric analysis of complement fragments on platelet MV in patient plasma confirmed dose-dependent complement activation by ATG. However, the rapid rise in MV numbers observed in vitro was not seen during ATG treatment. In vitro experiments suggested that this was due to adherence of C3b-tagged MV to red blood cells via complement receptor CR1. These data suggest a clinically relevant link between complement activation and thrombin generation and offer a potential mechanism underlying ATG-induced hypercoagulability.

Schistosoma mansoni tetraspanning orphan receptor (SmTOR): a new vaccine candidate against schistosomiasis.

One approach to fight against schistosomiasis is to develop an efficient vaccine. Schistosoma mansoni tetraspanning orphan receptor (SmTOR) might be a vaccine candidate, as it is a tegument membrane protein expressed most highly in cercariae. In this study we characterized the recombinant first extracellular domain of SmTOR (rSmTORed1) as having the expected property to bind C2 of complement similarly to a smaller peptide of the same domain, and to produce specific and high-titre antibodies in BALB/c mice immunized using complete Freund's adjuvant/incomplete Freund's adjuvant (CFA/IFA). Immunization was protective against parasite infection, as demonstrated by a significant decrease in worm burden in immunized BALB/c mice versus the control groups over two independent trials [64 and 45% reduction for mean adult worm burden in immunized versus phosphate-bufferd saline (PBS) injected mice]. Interestingly, infection by itself did not lead to the generation of anti-rSmTORed1 antibodies, corresponding to the low frequency of specific anti-rSmTORed1 antibodies detected in the sera of patients infected with S. mansoni (2/20; 10%). These data suggest that, as opposed to the natural infection during which SmTOR induces antibodies only rarely, immunization with its smaller first extracellular domain might be more efficient.

Ectosomes as modulators of inflammation and immunity.

Vesicles released by cells have been described using various names, including exosomes, microparticles, microvesicles and ectosomes. Here we propose to differentiate clearly between ectosomes and exosomes according to their formation and release. Whereas exosomes are formed in multi-vesicular bodies, ectosomes are vesicles budding directly from the cell surface. Depending upon the proteins expressed, exosomes activate or inhibit the immune system. One of the major properties of exosomes released by antigen-presenting cells is to induce antigen-specific T cell activation. Thus, they have been used for tumour immunotherapy. By contrast, the major characteristics of ectosomes released by various cells, including tumour cells, polymorphonuclear leucocytes and erythrocytes, are the expression of phosphatidylserine and to have anti-inflammatory/immunosuppressive activities similarly to apoptotic cells.

Identification of membrane-bound CR1 (CD35) in human urine: evidence for its release by glomerular podocytes.

Complement receptor 1 (CR1) is present on erythrocytes (E-CR1), various leucocytes, and renal glomerular epithelial cells (podocytes). In addition, plasma contains a soluble form of CR1 (sCR1). By using a specific ELISA, CR1 was detected in the urine (uCR1) of normal individuals (excretion rate in 12 subjects, 3.12 +/- 1.15 micrograms/24 h). Contrary to sCR1, uCR1 was pelleted by centrifugation at 200,000 g for 60 min. Analysis by sucrose density gradient ultracentrifugation showed that uCR1 was sedimenting in fractions larger than 19 S, whereas sCR1 was found as expected in fractions smaller than 19 S. The addition of detergents reduced the apparent size of uCR1 to that of sCR1. After gel filtration on Sephacryl-300 of normal urine, the fractions containing uCR1 were found to be enriched in cholesterol and phospholipids. The membrane-association of uCR1 was demonstrated by analyzing immunoaffinity purified uCR1 by electron microscopy which revealed membrane-bound vesicles. The apparent molecular mass of uCR1 was 15 kD larger than E-CR1 and sCR1 when assessed by SDS-PAGE and immunoblotting. This difference in size could not be explained on the basis of glycosylation only, since pretreatment with N-glycosidase F reduced the size of all forms of CR1; however, the difference in regular molecular mass was not abrogated. The structural alleles described for E-CR1 were also found for uCR1. The urine of patients who had undergone renal transplantation contained alleles of uCR1 which were discordant with E-CR1 in 7 of 11 individuals, indicating that uCR1 originated from the kidney. uCR1 was shown to bind C3b-coated immune complexes, suggesting that the function of CR1 was not destroyed in urine. A decrease in uCR1 excretion was observed in 3 of 10 patients with systemic lupus erythematosus, corresponding to the three who had severe proliferative nephritis, and in three of three patients with focal sclerosis, but not in six other patients with proteinuria. Taken together, these data suggest that glomerular podocytes release CR1-coated vesicles into the urine. The function of this release remains to be defined, but it may be used as a marker for podocyte injury.

Murine clusterin: molecular cloning and mRNA localization of a gene associated with epithelial differentiation processes during embryogenesis.

Clusterin is a broadly distributed glycoprotein constitutively expressed by various tissues and cell types, that has been shown to be involved in cell-cell adhesion and expressed during cellular differentiation in vitro. To assess the suggested participation of clusterin in these processes in vivo, we have cloned the cDNA encoding murine clusterin and studied the cellular distribution of clusterin mRNA during murine embryogenesis. Sequence analysis of the cDNA encoding murine clusterin revealed 92 and 75% sequence identity with the rat and human cDNAs, respectively, and conservation of the predicted structural features which include alpha-helical regions and heparin-binding domains. From 12.5 d of development onwards, the clusterin gene is widely expressed in developing epithelia, and selectively localized within the differentiating cell layers of tissues such as the developing skin, tooth, and duodenum where proliferating and differentiating compartments are readily distinguished. In addition, transient and localized clusterin gene expression was detected in certain morphogenetically active epithelia. In the lung, abundant gene transcripts were detected in cuboidal epithelial cells of the terminal lung buds during branching morphogenesis, and in the kidney, clusterin gene expression in the epithelial cells of comma and S-shaped bodies coincided with the process of polarization. Our results demonstrate the in vivo expression of the clusterin gene by differentiating epithelial cells during murine embryogenesis, and provide novel evidence suggesting that clusterin may be involved in the differentiation and morphogenesis of certain epithelia.

Schistosoma mansoni TOR is a tetraspanning orphan receptor on the parasite surface.

SUMMARY: A trispanning orphan receptor (TOR) has been described in Schistosoma haematobium and S. mansoni. Here we report the complete molecular organization of the S. mansoni TOR gene, also known as SmCRIT (complement C2 receptor inhibitor trispanning). The SmTOR gene consists of 4 exons and 3 introns as shown by cloning the single exons from S. mansoni genomic DNA and the corresponding cDNA from the larval stage (cercaria) and the adult worm. The SmTOR ORF consists of 1260 bp and is longer than previously reported, with a fourth trans-membrane domain (proposed new name: Tetraspanning Orphan Receptor) and with, however, an unchanged C2-binding domain on the extracellular domain 1 (ed1). This domain differs in S. japonicum. A protein at the approximate expected molecular weight (55 kDa) was detected in adult worm extracts with polyclonal and monoclonal antibodies, and was found to be expressed on the tegumental surface of cercariae.

Distinct sites of production and deposition of the putative cell death marker clusterin in the human thymus.

Clusterin is a multifunctional protein endowed with cell-aggregating, complement-inhibitory, and lipid-binding properties. Since several studies have demonstrated highly increased clusterin gene expression in epithelial and nervous tissues regressing as a consequence of tissue involution and apoptotic cell death, clusterin is also considered as a specific marker of dying cells. To determine whether clusterin expression is also upregulated during thymocyte death occurring during the negative selection process we analyzed the cellular distribution of clusterin mRNA and protein by in situ hybridization and immunocytochemistry in the human thymus. We observed that the expression of clusterin mRNA was confined to cells present in the thymic medulla, concentrated mainly around Hassal's bodies. Immunostaining of adjacent sections with antikeratin Ab revealed that cells containing clusterin mRNA were predominantly epithelial. By contrast no clusterin mRNA was found in thymocytes by in situ hybridization and Northern blot analysis of total RNA from purified thymocyte populations. Clusterin protein colocalized with the membrane attack complex of complement and vitronectin in the center of the largest Hassal's bodies, but was not detectable by immunocytochemistry in or at the surface of epithelial cells. Our results demonstrate that clusterin gene expression does not take place in apoptotic thymocytes, and therefore that clusterin synthesis by the dying cell is probably not a prerequisite to its death. However, synthesis of clusterin by medullary epithelial cells may be related to their terminal differentiation, and, furthermore, its presence in Hassal's bodies raises the possibility that the secreted protein is involved in the disposal of cell debris resulting from thymocyte apoptosis.

Human clusterin gene expression is confined to surviving cells during in vitro programmed cell death.

Clusterin is a serum glycoprotein endowed with cell aggregating, complement inhibitory, and lipid binding properties, and is also considered as a specific marker of dying cells, its expression being increased in various tissues undergoing programmed cell death (PCD). However, no study has so far directly shown that cells expressing clusterin in these tissues are actually apoptotic as defined by morphological and biochemical criteria. We have studied cellular clusterin gene expression in vitro using three different models of PCD: (a) ultraviolet B (UV-B) irradiation of human U937, HeLa, and A431 cell lines, (b) in vitro aging of human peripheral blood neutrophils (PMNs), and (c) dexamethasone-induced cell death of the human lymphoblastoid cell line CEM-C7. In all three models, the classical morphological and biochemical features of PCD observed did not correlate with an increase, but with either a marked decrease or an absence of clusterin gene expression as assessed by Northern blot analysis. In situ hybridization of U937 and A431 cells after UV-B irradiation revealed, in addition, that only morphologically normal cells that are surviving continue to express the clusterin gene. Our results demonstrate that in the human myeloid, lymphoid, and epithelial cell types studied, clusterin gene expression is not a prerequisite to their death by apoptosis. In addition, and most interestingly, in situ hybridization of U937 and A431 cells revealed that only surviving cells express the clusterin gene after the induction of PCD, thus providing novel evidence suggesting that clusterin may be associated with cell survival within tissues regressing as a consequence of PCD.

Formation of soluble immune complexes by complement in sera of patients with various hypocomplementemic states. Difference between inhibition of immune precipitation and solubilization.

To examine whether the ability of complement to form soluble immune complexes plays a role in preventing immune complex-mediated diseases, we analyzed the capacity of complement to inhibit immune precipitation (IIP) and to solubilize preformed immune aggregates (SOL) in 23 sera of patients with various hypocomplementemic states, and we correlated the results of these studies with the clinical syndromes found in the various patients. In sera with deficiency or depletion of early classical pathway components, IIP was profoundly altered, whereas SOL was delayed but in the normal range. In contrast, in sera with C3 depletion but intact classical pathway and in properdin-deficient serum, IIP was initially preserved, whereas SOL was abolished. Since the incidence of immune complex diseases in various hypocomplementemic states correlates with the severity of IIP defects, but not with reduced SOL, it is suggested that IIP is an essential biological function of complement that prevents the rapid formation of insoluble immune complexes in vivo.

Soluble complement receptor 1 is increased in patients with leukemia and after administration of granulocyte colony-stimulating factor.

Complement receptor type 1 is expressed by erythrocytes and most leukocytes. A soluble form is shed from the leukocytes and found in plasma (sCR1). sCR1 is a powerful inhibitor of complement. We report an increased sCR1 in the plasma of leukemia patients, up to levels producing measurable complement inhibition. Half of the 180 patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL) had sCR1 levels above the normal range. The highest levels were observed in T-ALL (17 patients). The complement function of a T-ALL serum was improved by blocking sCR1 with a specific mAb (3D9). Measurements in 16 peripheral stein cell donors before and after granulocyte colony-stimulating factor (G-CSF) administration showed an increase in sCR1 (before, 43.8+/-15.4; at day 5, 118.3+/-44.7 ng/mL; P < 0.0001). This increase paralleled the increase in total leukocyte counts and was concomitant with de novo leukocyte mRNA CR1 expression in all three individuals tested. Whether pharmacological intervention may be used to up-regulate sCR1 so as to inhibit complement in vivo should be further investigated.

[Systemic lupus erythematosus--myths and dogma]. / Systemischer Lupus Erythematodes--Mythos und Dogma.

In this short review, four different aspects of SLE are discussed. The ACR criteria for SLE were established for the differentiation of SLE from other autoimmune diseases and not for the direct diagnosis of SLE. Treatment of SLE is continuously re-evaluated thanks ongoing clinical research. Best clinical practice should be based on experienced and continuous knowledge in the field rather than on dogmatic treatment schemes. SLE is not one disease, but a series of clinical pictures associated into a syndrome. Finally, do not forget that patients with the SLE syndrome suffer almost always from debilitating fatigue.

Complement activation by pulsed tunable dye laser in normal skin and hemangioma.

Pulsed tunable dye laser (577 nm) (PTDL) therapy induces hemoglobin coagulation and tissue necrosis, which is mainly limited to blood vessels. To define whether this treatment activates complement in normal skin and senile hemangioma, we analyzed complement deposition in blood vessels by immunofluorescence. C3 fragments, C8, and C9 were detected with specific polyclonal antibodies. The membrane attack complex of complement (MAC) was demonstrated with a monoclonal antibody which reacts only with a neoantigen of MAC. Amplification of C3 deposition by the alternative pathway was determined on cryostat sections by indirect immunofluorescence with use of C4 deficient guinea pig (GP) serum. Normal skin and hemangiomas from three individuals were studied. In PTLD-irradiated normal skin, the main findings were as follows: 1) C3 fragments, C8, C9, and MAC were deposited in vessel walls; 2) these deposits were not due to denaturation of the proteins since they became apparent only 7 min after irradiation, contrary to immediate deposition of transferrin at the sites of erythrocyte coagulates; 3) the C3 deposits were shown to amplify complement activation by the alternative pathway, a reaction which was specific since tissue necrosis itself did not lead to such amplification; 4) these reactions preceded the local accumulation of polymorphonuclear leucocytes. Tissue necrosis was more pronounced in the hemangiomas. The larger angiomatous vessels in the center of the necrosis did not fix complement significantly. By contrast, complement deposition in the vessels situated at the periphery was similar to that observed in normal skin with one exception: C8, C9, and MAC were detected in some blood vessels immediately after laser treatment, a finding consistent with assembly of the MAC occurring directly without the formation of a C5 convertase. These results indicate that complement is activated in PTDL-induced vascular necrosis, and might be responsible for the ensuing inflammatory response.

Membrane attack complex (MAC) deposits in skin are not always accompanied by S-protein and clusterin.

Clusterin and S-protein bind to the membrane attack complex of complement (MAC) rendering it cytolytically inactive. Tissue necrosis as produced by pulsed tunable dye laser therapy (PTDL), and immune complex-related diseases such as lupus erythematosus, are accompanied by local accumulation of MAC. However, the mechanisms responsible for this accumulation might differ, and lead to deposition of MAC in different forms (cytolytically active or inactive). Biopsy specimens of lesional (22) and non-lesional (10) skin from 27 patients with a positive lupus band test (LBT) were studied using monoclonal antibodies against clusterin, S-protein, and MAC by immunofluorescence and immunoperoxidase. Identical studies were performed in normal and angiomatous skin specimens from three normal individuals before and after laser irradiation. MAC was present in 30 of 32 positive LBT skin biopsies. MAC was not only present in lesional (21 of 22) but also in non-lesional skin (nine of 10), although the intensity of staining appeared to be lower in the latter. Clusterin and S-protein co-localized with MAC, respectively, in 20 and 12 specimens, and were not found in the absence of MAC. In addition S-protein deposits were seen only in biopsies positive for clusterin. Deposits of clusterin and S-protein did not correlate with the presence or absence of lesions. After irradiation with PTDL, the immediate complement activation was accompanied by MAC deposits that were granular and clearly located on vascular endothelial cells. Clusterin and S-protein were not present on these cells. In summary, clusterin localizes with MAC along the skin dermal-epidermal junction in patients with a positive LBT, suggesting that it has a similar and possibly more important role than S-protein in regulating immune complex-mediated MAC formation. By contrast, clusterin and S-protein are not involved at the time of MAC formation in cells undergoing necrosis after PTDL therapy.

Activation of the alternative pathway of complement by skin immune deposits.

Skin immune deposits at the basement membrane zone have been demonstrated by functional assays to activate complement. This important biologic function has not yet been explored for immune deposits present in other locations mainly because many cytoplasmic structures in the skin have the capacity to activate the complement cascade by the classical pathway. In this study the capacity of immune deposits to activate directly the alternative pathway was examined using a functional guinea pig C3 binding test. This test was devised so as to avoid complement activation by normal cutaneous structures, thus it did not examine the capacity of immune reactants to activate the classical pathway. The main findings were that alternative pathway activation could be demonstrated only when human C3 deposits were seen by direct immunofluorescence, but not all C3 deposits were found to activate the alternative pathway; such activation was restricted to vascular deposits; the phlogistic potential of the immune deposits correlated with serologic evidence of ongoing immune reactions, i.e., hypocomplementemia and circulating immune complexes. It is suggested that this test provides data on one aspect of the phlogistic potential of skin immune deposits not detectable by direct immunofluorescence.

Clusterin gene expression mediates resistance to apoptotic cell death induced by heat shock and oxidative stress.

Clusterin is a widely expressed, well conserved, secreted glycoprotein, which is highly induced in tissues regressing as a consequence of apoptotic cell death in vivo. It has recently been shown that clusterin expression is only confined to surviving cells following the induction of apoptosis in vitro, suggesting that it is involved in cell survival rather than death. In the hypothesis that clusterin may be implicated in cellular responses to stress, clusterin gene expression was analyzed in the A431 human epidermoid cancer cell line following heat shock and oxidative stress. Our results show that both a transient heat shock (20 min at 42 degrees C) and various oxidative stresses, including hydrogen peroxide, superoxide anion, hyperoxia and UVA exposure, induce a strong increase in clusterin mRNA levels as assessed by northern blot. Nuclear run-on analysis suggests that transcriptional activation is involved in inducing clusterin mRNA in response to heat shock. Using pulse-chase analysis of control and heat shocked cells, it is shown that clusterin mRNA is translated and secreted, thus resulting in increased extracellular levels of the protein following heat shock. To investigate the function of clusterin in response to these stresses, clusterin anti-sense transfectants that stably express virtually no clusterin at the mRNA and protein level were generated in A431 cells. These anti-sense transfectants are shown to be highly sensitive to apoptotic cell death induced by heat shock or oxidative stress compared with wild-type A431 cells or control transfectants. Taken together, our results show that clusterin gene expression is induced in response to heat shock and oxidative stress in human A431 cells, and confers cellular protection against heat shock and oxidative stress.

Paraproteins and complement depletion: pathogenesis and clinical syndromes.

Various clinical syndromes that associate paraproteinemia and complement depletion have been described in the last three decades. Among these, cryoglobulinemias, acquired Clq deficiency, and acquired deficiencies of the classical pathway of complement can be associated with B-cell lymphoproliferative disorders. Some specific symptoms should alert the clinician to suspect an underlying malignancy. In this report, we review the pathogenesis, symptomatology and therapeutic options of these clinical conditions.

Complement-mediated adherence of immune complexes to human erythrocytes. Difference in the requirements for C4A and C4B.

The classical pathway of complement is required for the adherence of soluble tetanus toxoid (TT)-human anti-TT complexes to erythrocytes. Using human C4-deficient serum we compared the capacity of the two forms of human C4 (C4A and C4B) to mediate this function: C4A was shown to be 1.5-fold more efficient than C4B. In contrast, haemolysis by C4B was 3.7-fold more efficient than by C4A. Such large differences suggest that both forms are complementary, and that C4A is preferentially involved in the processing of immune complexes in humans.

Solubilization of immune precipitates by complement in the absence of properdin or factor D.

Various experiments have demonstrated that immune precipitates (IPs) are not solubilized by complement in the absence of alternative pathway function. To determine whether the characteristics of the IPs were responsible for these observations, we studied the solubilization (Sol) of IPs formed by bovine serum albumin (BSA)-rabbit antiBSA and tetanus toxoid (TT)-human antiTT. Sera deficient in properdin solubilized a fraction of BSA-antiBSA precipitates, although only when the IPs were formed in antibody excess. The same sera solubilized TT-antiTT precipitates with some delay but almost as efficiently as normal serum. Factor D-depleted serum solubilized a fraction of TT-antiTT precipitates too, indicating that Sol may proceed through activation of the classical pathway only. Thus, the requirements for complement-mediated Sol depend on the characteristics of the IPs and do not necessarily include alternative pathway function.

A simple two-step procedure for the preparation of the first component of human complement (C1) in its native form.

Native human C1 was purified from fresh human serum by affinity chromatography on protein A-bound Sepharose in the presence of 4-nitrophenyl-4-guanidinobenzoate hydrochloride (NPGB) taking advantage of the successive binding of IgG to protein A followed by C1 binding to IgG. After elution the C1 preparation contained IgG as a major contaminant as shown by SDS-PAGE. C1 was further purified by gel filtration. The yield of C1 was 12% and less than 4% of this C1 was activated during purification as assessed by a C4 consumption assay.

Purification of human complement factor D from the peritoneal fluid of patients on chronic ambulatory peritoneal dialysis.

We describe a simple procedure for the purification of human factor D from the peritoneal fluid (PF) of patients with end stage renal failure (ESRF) on chronic ambulatory peritoneal dialysis (CAPD). The main advantages of this method are: (1) a relative enrichment of factor D in PF versus plasma (factor D/total protein enriched 3.8-fold); thus, added to the elevated concentration of factor D in ESRF, the enrichment compared to normal human serum is approximately 50 fold. (2) This biological source of factor D is almost unlimited, since around 10 liters of PF are removed per day from each patient. The purification is performed in three simple steps-Bio Rex 70, Heparin Sepharose CL 6B and Mono S FPLC- and milligrams of highly purified factor D are obtained. Peritoneal fluid might be a valuable source for the purification of other low MW proteins which accumulate in renal failure.