Search PubMed⌕ Search

Biomedical subjects

J P Paccaud

Publications and source records attributed to J P Paccaud.

At least 19 recordsLinked to original sources
PubMed1992

Internalization of the human insulin receptor. The insulin-independent pathway.

Internalization of the human insulin receptor requires the activation by insulin of the intrinsic kinase of the receptor. However, even in the absence of kinase activation, insulin receptors slowly enter the cells. In the present study, we addressed the question of this insulin-independent pathway of internalization. To that end, we traced insulin receptor internalization with a monoclonal antibody (mAb 83-14) directed against the alpha-subunit of the human insulin receptor. Internalization of this antibody was followed in Chinese hamster ovary (CHO) cells transfected with either normal (CHO.HIRC2) or kinase-deficient (CHO.A1018) human insulin receptors. The internalization rate of 125I-mAb 83-14 was comparable in CHO cells expressing kinase-active or kinase-inactive receptors and was similar to that observed for 125I-insulin in CHO.A1018 cells. Moreover, in CHO.HIRC2 cells, the internalization of 125I-mAb 83-14 was identical with that of its 125I-Fab fragments. Thus, mAb 83-14 represents an appropriate tool to study the constitutive internalization of the insulin receptor. Internalization of insulin receptors tagged with 125I-mAb 83-14 was unaffected by cytochalasin B, which excluded a macropinocytotic process. By contrast, internalization was sensitive to hypertonia, which abrogates clathrin-coated pits-mediated endocytosis. The implication of clathrin-coated pits in this internalization process was directly demonstrated by quantitative electron microscopic autoradiography, which showed that 125I-mAb 83-14 present on the nonvillous domain of the cell surface preferentially associate with clathrin-coated pits at all time points.

Animals↗
PubMed1992

Insulin-induced surface redistribution regulates internalization of the insulin receptor and requires its autophosphorylation.

The role of insulin-induced receptor autophosphorylation in its internalization was analyzed by comparing 125I-labeled insulin (125I-insulin) internalization in Chinese hamster ovary (CHO) cell lines transfected with normal (CHO.T) or mutated insulin receptors. In four cell lines with a defect of insulin-induced autophosphorylation, 125I-insulin internalization was impaired. By contrast, in CHO.T cells and in two other CHO cell lines with amino acid deletions or insertions that do not perturb autophosphorylation, 125I-insulin internalization was not affected. A morphological analysis showed that the inhibition is linked to the ligand-specific surface redistribution in which the insulin-receptor complexes leave microvilli and concentrate on nonvillous segments of the membrane where endocytosis occurs.

Animals↗
PubMed1991

Internalization pathway of C3b receptors in human neutrophils and its transmodulation by chemoattractant receptors stimulation.

On the surface of phagocytes, C3b receptors (CR1) bind C3b-coated particles and promote their ingestion after activation by appropriate stimuli such as lymphokines or the chemoattractant formyl methionyl leucyl phenylalanine (fMLP) and fibronectin. The aims of the present study were 1) to define at the electron microscopic level the nature of the process responsible for CR1 internalization and 2) to dissect the mechanism by which a physiological activator (fMLP) stimulates this process. CR1 was visualized either by the immunogold technique or by quantitative electron microscopic autoradiography using a monoclonal anti-CR1 antibody. Both techniques revealed that after anti-CR1 binding, CR1 cluster on the neutrophil surface in a time-, temperature-, and antibody-dependent fashion, but do not concentrate in coated pits. CR1 internalization requires receptor cross-linking (does not occur in the presence of Fab fragments of anti-CR1) and intact microfilaments. It results in the association of the internalized material with large flattened vacuoles, organized in stacks. Together with the surface localization of CR1 close to cytoplasmic projections (ruffles), these observations suggest that uptake of CR1 occurs through a macropinocytotic process. Eventually, CR1 concentrate in lysosomal structures. fMLP markedly stimulates this pattern of CR1 internalization without affecting their clustering or their lack of association with coated pits. Stimulation by fMLP is inhibited by pertussis toxin, unaffected by preventing receptor-triggered cytosolic free calcium [Ca2+]i elevations, and mimicked by phorbol myristate acetate. Taken together our data demonstrate 1) that, in neutrophils, CR1 is internalized via a coated pit independent macropinocytotic process, dependent on intact microfilaments and receptor cross-linking; 2) that, in the same cells, fMLP is internalized via the classical coated pits pathway; and 3) that fMLP amplifies CR1 uptake possibly via protein kinase C stimulation.

Autoradiography↗
PubMed1991

Immune complex binding efficiency of erythrocyte complement receptor 1 (CR1).

C3b-coated immune complexes adhere to the complement receptor 1 (CR1, CD35) on human erythrocytes. This multi-valent binding might be favoured by the known clustering of CR1 and by the multiple C3b-binding sites on each CR1. The size of the CR1 clusters correlates directly with the number of CR1/erythrocytes, and the different structural CR1 alleles bear between two and five C3b-binding sites. Using radiolabelled hepatitis B surface antigen-antibody complexes, we investigated whether CR1 numbers and structural alleles modulate the ability of erythrocytes to bind immune complexes, and assessed if any reorganization of immune complexes takes place at the erythrocyte surface after the initial binding reaction. The binding efficiency (immune complexes/CR1) correlated with CR1 number as determined by the maximal binding at 4 degrees C, the kinetics of binding at 37 degrees C, and the binding in the presence of excess immune complexes and of immune complexes of small size. Binding efficiencies were similar for erythrocytes with low CR1 from normal subjects and patients with AIDS or SLE. A monoclonal antibody blocking the C3b-binding sites (3D9) of CR1 interfered with binding efficiency at a lower concentration on cells bearing low CR1 numbers, suggesting that CR1 clustering is essential. The larger alleles of CR1 (DD and BB) were more efficient than AA alleles. The distribution of immune complexes, visualized by immunofluorescence, was heterogeneous on erythrocytes: about two out of three cells bore between one and 12 immune complexes. No visible immune complex reorganization took place after initial binding, as prefixed erythrocytes displayed the same immune complex distribution and number/erythrocytes as unfixed erythrocytes. The contribution of CR1 alleles in immune complex binding efficiency was confirmed by morphological analysis. These results demonstrate that immune adherence efficiency is the resultant of the CR1 clustering, as well as the particular alleles carried by erythrocytes. Moreover, there is little or no immune complexes surface reorganization after the initial binding reaction.

Antibodies, Monoclonal↗
PubMed1990

Exudation induces clustering of CR1 receptors at the surface of human polymorphonuclear leukocytes.

The complement receptor type 1 (CR1) surface distribution, density and immune adherence efficiency were determined in circulating PMN activated by fMLP, NAP-1/IL-8, TNF, GM-CSF and C5a, or exudate PMN harvested from skin-blisters. These observations were compared with those observed on resting peri-pheral blood PMN. PMN activators known to upregulate CR1 expression did not induce a significant increase in CR1 clustering, or immune adherence efficiency towards opsonized immune complexes. By contrast, increase in CR1 density at the surface of exudated PMN was accompanied by an increased clustering. This clustering was however insufficient to increase the binding efficiency for immune complexes. Eventually, CR1 expression of exudated neutrophil could not be increased further by stimulation with fMLP or PMA. These results indicated that clustering of CR1 on PMN may occur in vivo. Such reaction might determine the phagocytic potential of the cell for opsonized micro-organisms or debris. This clustering could not be attributed to one of the PMN activators tested.

Complement C1↗
PubMed1990

NAP-1/IL-8 induces up-regulation of CR1 receptors in human neutrophil leukocytes.

The effect of the neutrophil-activating peptide NAP-1/IL-8 on the expression of complement receptor type 1 (CR1) in human neutrophils was studied. NAP-1/IL-8 enhanced CR1 expression at concentrations between 10(-10) and 10(-8) M. The maximum increase with respect to unstimulated control cells was on average 2.3 fold. The effect was rapid: Half-maximum enhancement was obtained in 4 min and the plateau was reached in 15 min. The chemotactic peptide fMLP, tested for comparison, was effective between 10(-9) and 10(-7) M, showed a similar time course and a somewhat higher maximum effect (2.8 fold increase). The effect of NAP-1/IL-8 was prevented by pretreatment of the cells with B.pertussis toxin and desensitization was observed following restimulation. Stimulus combination experiments suggested that NAP-1/IL-8 mobilizes the same or a similar intracellular pool of CR1 receptors as fMLP or C5a.

Chemotactic Factors↗
PubMed1990

Difference in the clustering of complement receptor type 1 (CR1) on polymorphonuclear leukocytes and erythrocytes: effect on immune adherence.

Complement receptor type 1 (CR1) mediates the adherence of complement-reacted immune complexes (IC) to various blood cells. On the erythrocyte, CR1 are clustered, a distribution which favors efficient multivalent binding of C3b-coated IC. IC can also bind to CR1 expressed on polymorphonuclear (PMN) leukocytes. To evaluate the respective importance of these two cell types in immune adherence reactions, functional analysis of IC binding, as well as morphological studies of CR1 distribution at their surface were undertaken. At equal cell concentrations, resting PMN leukocytes bound the same percentage of IC as erythrocytes, despite expressing four times more CR1 at their surface. At equal CR1 concentrations, IC binding to resting or formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN leukocytes was always lower than to erythrocytes. The morphological counterpart of these differences was studied by label-fracture immunoelectron microscopy. On erythrocytes, almost 50% of the CR1 were distributed in clusters of greater than or equal to 3 units, while less than 15% were grouped in such clusters on the surface of PMN leukocytes. Activation of PMN leukocytes by fMLP increased the surface density of CR1, but the proportion of clustered CR1 remained unchanged. These observations suggest that the low responsiveness of PMN leukocytes towards C3b-coated IC may be due to the unaggregated state of CR1. In the circulation, erythrocytes might function as a "buffer" for PMN leukocytes, which would otherwise engage too swiftly in reactions with IC.

Antigen-Antibody Complex↗
PubMed1989

Two isotypes of human C4, C4A and C4B have different structure and function.

The two types of human C4, C4A and C4B, differ in their amino acid sequence and in their capacity to bind to different acceptor sites. C4B is more efficient than C4A in haemolytic assays; by contrast C4A binds preferentially to immune complexes. In assays comparing haemolysis to processing of immune complexes the two types of C4 differ more than fivefold. Thus, the classical pathway is a duplicated system that allows the formation of a C3 convertase on various substrates: this duplication may be of vital importance to eliminate invading microorganisms. In addition, the clinical observation of an increased incidence of homozygous C4A null alleles in systemic lupus erythematosus may be explained in part by defective processing of immune complexes in the absence of C4A.

Antigen-Antibody Complex↗
PubMed1989

Reduced immune adherence of antigen/antibody complexes formed in the presence of complement in vivo and in vitro.

The adherence of complement-reacted immune complexes (ICs) to cells bearing C3b receptors depends on the characteristics of the IC used. The immune adherence of preformed ICs exposed in vitro or in vivo to complement has been well established, and was confirmed using different types of ICs-antigens used: BSA; BSA dimers and trimers; tetanus toxoid, and hepatitis B surface antigen. In contrast the same ICs did not bind to human red blood cells when formed in the presence of serum in vitro. ICs remained negative for immune adherence as well, when formed directly in vivo by the sequential injection of antibody and antigen in guinea pigs. These results suggest that in many circumstances, the elimination of ICs formed in the circulation does not involve immune adherence reactions, possibly because complement in itself inhibits the formation of the large ICs that would bind to C3b receptor-bearing cells.

Animals↗
PubMed1989

The role of hypocomplementaemia and low erythrocyte complement receptor type 1 numbers in determining abnormal immune complex clearance in humans.

Defective clearance of immune complexes (IC) may contribute to the pathogenesis of diseases such as SLE. We studied the effect of hypocomplementaemia and the influence of erythrocyte complement receptor type 1 (CR1, CD35) number on the clearance of radiolabelled tetanus toxoid (TT)-anti-TT IC from the circulation. These were injected intravenously into 9 normal subjects and 15 patients with diseases characterized by IC formation and/or hypocomplementemia, including 2 with hereditary complement deficiency. IC were found to bind to erythrocyte CR1 in a complement-dependent manner and their degree of uptake was directly correlated with CR1 numbers. Two phases of IC clearance were identified. The first was rapid, occurring within 1 min. Since this phase might represent inappropriate deposition of IC in target organs we called it trapping. It was seen predominantly in subjects with low CR1, low complement, and low binding of complexes to red cells. The second phase was monoexponential with a mean elimination rate of 14.1%/min; it was inversely correlated with CR1 numbers and binding of complexes to red cells. In a second study each individual was injected with IC bound to autologous erythrocytes in vitro using normal serum so that the effects of complement deficiency were eliminated. Up to 81.4% of these bound IC were released in vivo from erythrocytes in 1 min, and the proportion was inversely correlated with CR1 numbers. Only five patients showed trapping, and these had low CR1 numbers and high percentage release of IC. The second phase of elimination was inversely correlated with CR1 numbers and the proportion of IC remaining bound to red cells at 1 min. The two complement-deficient patients had normal CR1: when IC were injected, trapping and very fast clearance rates were observed; however complexes that had been opsonized and bound to erythrocytes were cleared at a slower rate without evidence for trapping. These studies show that complement and erythrocyte CR1 may determine the physiological clearance of certain types of IC and suggest that this system may function abnormally when CR1 number or complement function are reduced.

Antigen-Antibody Complex↗
PubMed1989

Complement activation by the alternative pathway is modified in renal failure: the role of factor D.

Factor D, an essential enzyme of the alternative pathway (AP) of complement, is eliminated by the kidney, and its plasma concentration increases 10-fold in end-stage renal disease (ESRD). The purpose of this study was to analyze the consequences of factor D accumulation. A number of in vitro assays were used to analyze AP activation in normal human serum (NHS), in normal serum supplemented with purified factor D to 10-fold its normal concentration (10 x D), and in sera of patients with ESRD. When compared with NHS, in 10 x D: 1) Spontaneous fluid-phase activation of complement at 37 degrees C was greatly increased as measured by C3 cleavage, 2) The lysis of rabbit erythrocytes, a function of the AP, was accelerated, 3) More C3 fragments bound to cuprophane membranes and to immune precipitates; both reactions were accompanied by the formation of more C5a, 4) Complement mediated solubilization of antigen-antibody precipitates was enhanced. Sera of patients with ESRD behaved similarly to 10 x D in all assays used, i.e., enhanced AP function, although complement activation measured in these assays varied widely from one individual to another. Thus, the elevated factor D concentration observed in renal failure might have important pathophysiological consequences, some of which could be detrimental (e.g., C5a produced during hemodialysis), while others might be beneficial, e.g., solubilization of immune precipitates.

Aged↗
PubMed1988

Direct evidence for the clustered nature of complement receptors type 1 on the erythrocyte membrane.

C receptors 1 (CR1) of human E are involved in the transport of C3b-coated immune complexes (IC) in the circulation. Many studies have suggested that the binding of IC to E is multivalent. This would require CR1 to be clustered on the cell membrane, but no direct evidence for such clustering is available. We studied the distribution of CR1 on human E by immunofluorescence and shadow-casting immuno-electron microscopy techniques with the use of a monoclonal anti-CR1 antibody followed by FITC- or gold-conjugated second antibodies, respectively. By immunofluorescence, CR1 appeared as small dots (clusters) on fixed and unfixed E prepared either at 4 degrees C or at 37 degrees C. In the same donor, the number of clusters varied extensively from cell to cell (e.g., 1 to 43 clusters/E for a donor with 520 CR1/cell), but the mean number of clusters per cell correlated significantly with the mean number of CR1/cell. These images contrasted with those obtained for Rhesus D (RhD) Ag used as controls (RhD Ag are known to be evenly distributed): only a faint uniform fluorescence was seen despite the presence of 10,000 antigenic sites. As determined by immunocytochemical method, more than 65% of the total gold particles were organized in clusters (2 to 15 gold particles/cluster) whether cells were prefixed or not. Quantitative determinations suggested that each gold particle corresponded to one CR1. The fraction of gold particles grouped into clusters of three or more receptors, the mean size of the clusters, and the maximal size of clusters correlated with the mean number of CR1 per cell. By contrast, RhD Ag were distributed homogeneously (less than 2% gold particles in clusters). These data are the first to demonstrate the preclustered nature of CR1 on E. Such distribution could explain the high binding efficiency of C3b-coated IC to E despite the low number of CR1 per cell.

Antibodies, Monoclonal↗
PubMed1988

Macro-prostatic acid phosphatase in a patient's serum.

A patient without prostatic carcinoma had a high concentration of prostatic acid phosphatase (PAP; EC 3.1.3.2) in his serum. This PAP was bound to IgG ("macro-PAP"), and IgG autoantibodies against PAP were demonstrated in serum. The patient's IgG prolonged the biological half-life of radiolabeled PAP in rats, suggesting that the formation of IgG-PAP complexes was responsible for decreased PAP catabolism. Furthermore, macro-PAP was inactivated in serum more slowly than PAP. These factors accounted for the increases in the enzymatic activity and antigenic concentration of PAP measured in the patient's serum. Inappropriate therapy was prescribed on the basis of this laboratory result. The diagnosis of prostatic carcinoma requires clinical or histological evidence of malignant disease, and should not rely solely on PAP measurements.

Acid Phosphatase↗
PubMed1987

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.

Antibodies↗
PubMed1987

Tetanus toxoid-anti-tetanus toxoid complexes: a potential model to study the complement transport system for immune complex in humans.

Complement and its receptor on erythrocytes appears to play a physiological role in the elimination of large immune complexes (IC) in monkeys, and a similar system is likely to work in humans. Here we define a safe IC model which is suitable for clinical investigations. Soluble tetanus toxoid (TT)-human anti-TT (IgG) antibody complexes were prepared in large antibody excess. The size of the complexes was approximately 45 S. When incubated in normal human serum, 50% of the IC increased further in size, but remained soluble, and bound rapidly to human erythrocytes in vitro. This binding was shown to require intact classical pathway function. When injected into normal guinea-pigs a comparable proportion of IC bound immediately to blood cells (mainly to platelets). No platelet binding of IC occurred in C4-deficient guinea-pigs, but this binding was restored when C4 was supplied. Initial immune complex elimination was faster in C4 deficient than in C4-supplemented and normal guinea pigs. Thus classical pathway function appeared to be necessary for the normal processing, transport and elimination of TT-anti-TT complexes.

Animals↗
PubMed1986

Difference in the biological properties of the two forms of the fourth component of human complement (C4).

The two forms of human C4 were compared in their haemolytic activity and in their capacity to mediate inhibition of immune complex precipitation in human C4 deficient sera. Whereas haemolysis by C4B was 3.2 fold more efficient than by C4A, C4A was 1.7 fold more efficient at inhibiting immune precipitation than C4B. Thus the biological properties of the two forms of human C4 are different and it is suggested that C4A is mainly involved in immune complex clearance reactions.

Antigen-Antibody Complex↗