Antiinflammatory effects of reconstituted high-density lipoprotein during human endotoxemia.

High-density lipoprotein (HDL) has been found to neutralize LPS activity in vitro and in animals in vivo. We sought to determine the effects of reconstituted HDL (rHDL) on LPS responsiveness in humans in a double-blind, randomized, placebo-controlled, cross-over study. rHDL, given as a 4-h infusion at 40 mg/kg starting 3.5 h before endotoxin challenge (4 ng/kg), reduced flu-like symptoms during endotoxemia, but did not influence the febrile response. rHDL potently reduced the endotoxin-induced release of TNF, IL-6, and IL-8, while only modestly attenuating the secretion of proinflammatory cytokine inhibitors IL-1ra, soluble TNF receptors and IL-10. In addition, rHDL attenuated LPS-induced changes in leukocyte counts and the enhanced expression of CD11b/CD18 on granulocytes. Importantly, rHDL infusion per se, before LPS administration, was associated with a downregulation of CD14, the main LPS receptor, on monocytes. This effect was biologically relevant, since monocytes isolated from rHDL-treated whole blood showed reduced expression of CD14 and diminished TNF production upon stimulation with LPS. These results suggest that rHDL may inhibit LPS effects in humans in vivo not only by binding and neutralizing LPS but also by reducing CD14 expression on monocytes.

Studies onthe chemical nature of lysine-binding sites and on their localization in human plasminogen.

The isolated 'kringle' structures 1 and 4 of human plasminogen lost lysine affinity upon photo-oxidation of histidine, but mostly retained it in the presence of 6-aminohexanoic acid. Lysine affinity was lost and could be partially restored after blocking of histidine with diethylpyrocarbonate and deblocking, or after esterification of COOH-groups and saponification. Only His-31 and most likely Asp-54 qualify as participants in a lysine binding site when the primary structures of the 'kringles' are considered.

Elevation of plasma high-density lipoprotein concentration reduces interleukin-1-induced expression of E-selectin in an in vivo model of acute inflammation.

BACKGROUND: Although there is strong evidence that plasma HDL levels correlate inversely with the incidence of coronary artery disease, the precise mechanism(s) for the protective effect of HDLs remains unclear. We recently showed that HDLs inhibit endothelial cell expression of cytokine-induced leukocyte adhesion molecules in vitro. Our study therefore sought to test the hypothesis that elevating the level of circulating HDLs would inhibit endothelial cell activation in vivo. METHODS AND RESULTS: We used a porcine model of inflammation previously established in our laboratory, in which the level of vascular endothelial cell expression of E-selectin in interleukin (IL)-1alpha-induced skin lesions was measured by the uptake of a radiolabeled anti-E-selectin antibody (1.2B6). Porcine plasma HDL levels were elevated by use of a bolus injection of reconstituted discoidal HDL (recHDL). These particles resemble nascent HDL particles in shape and contain apolipoprotein A-I as the sole protein and soybean phosphatidylcholine as the sole phospholipid. We found that recHDLs inhibited the expression of IL-1alpha-induced E-selectin by porcine aortic endothelial cells in vitro, confirming that the inhibitory effect is conserved with synthetic HDLs and demonstrating that the phenomenon is not restricted to human endothelial cells. In vivo, elevating the circulating level of HDLs approximately 2-fold led to significant inhibition of basal and IL-1alpha-induced E-selectin expression by porcine microvascular endothelial cells. CONCLUSIONS: These observations demonstrate the potential anti-inflammatory action of HDLs and provide support for the further investigation of the mechanisms underlying the inhibitory effects of HDLs on endothelial cell activation.

Monomeric and dimeric IgG1 as probes for assessing high-affinity and low-affinity receptors for IgG on human monocyte-derived macrophages and on activated macrophages.

From a panel of IgG1 myeloma proteins, only one was found to interact with human monocyte FcR in a manner similar to that of polyclonal IgG. This protein was used in binding studies involving human macrophage Fc receptors. A monomeric fraction depleted of dimeric and polymeric IgG1 was crosslinked with bis-diazonium benzidine, and a fraction highly enriched in cross linked IgG1 dimers was radiolabeled. Labeled monomeric and dimeric IgG were allowed to interact with monocytes that had matured to macrophages in vitro. The association with macrophages at 4 degrees C, in the presence of cytochalasin B, reached a plateau after 6 hr. The dissociation induced by excess unlabeled IgG followed similar kinetics as the association, but 20-30% of the bound IgG could not be dissociated. Under equilibrium conditions, evidence for a single FcR population binding monomeric IgG was obtained, the Kd being in the range of 12-42 nM. In contrast, the binding of dimeric IgG was more consistent with a model assuming two populations of binding sites when appropriate curve-fitting calculations were applied. The high-affinity FcR population had a Kd in the range of 0.8-3.5 nM, whereas the Kd of the low-affinity FcR population was in the range of 28-85 nM. When macrophages had been pre-treated with recombinant interferon-gamma, the expression of high-affinity sites was increased by a factor of 1.5-3, but the number of low-affinity sites was not augmented. Cytofluorographic analyses confirmed the increased expression of high-affinity FcR, binding fluoresceinating murine IgG2a. The expression of CD16, a low-affinity FcR expressed on neutrophils, NK cells and macrophages, as well as the expression of the complement receptor type III was little influenced by the rIFN-gamma pretreatment.

Isolation and purification of the human thymocyte antigens T6 and M241.

T6 and M241 antigens are products of the Class I major histocompatibility complex. The T6 and M241 antigens can be detected on human cortical thymocytes and on dendritic cells in the skin by monoclonal antibodies. Here we report a method of purification of the T6 and M241 antigens. Amino acid sequence data of purified antigens indicate that the heavy chains are blocked at their N-termini, whereas the partial N-terminal amino acid sequence of the light chains is identical to that of the human beta 2-microglobulin. In order to obtain sequence data from the heavy chains a method is described for isolation of purified cyanogen bromide fragments by electrophoretic methods.

Reconstituted high density lipoprotein (rHDL) modulates platelet activity in vitro and ex vivo.

A reconstituted high density lipoprotein (rHDL) prepared for clinical use was tested for its influence on platelet activity modulated by various stimuli. In a first series of in vitro experiments, rHDL was added to blood in a concentration series, and platelet rich plasma (PRP) was isolated. Platelets were stimulated with arachidonic acid, collagen, epinephrine or ADP, and platelet aggregation was assessed. rHDL mediated a dose dependent inhibition of the platelet activity. With purified platelets rHDL inhibited the release reaction induced by collagen, but not by thrombin, as measured by CD62P (P-Selectin) expression on the plasma membrane. Ex vivo experiments were performed with PRP from volunteers, previously infused with 25 mg rHDL/kg body weight and 40 mg rHDL/kg body weight, respectively. Platelet activity in PRP was assessed before, and up to 30 h after the end of the rHDL infusion. A transient inhibition of the platelet aggregation induced by arachidonic acid and collagen was observed which was more pronounced in the group receiving 40 mg rHDL/kg body weight. In both groups of experiments, in vitro and ex vivo, the inhibition of the platelet activity was also dependent on the stimulus used.

Differential effects of reconstituted high-density lipoprotein on coagulation, fibrinolysis and platelet activation during human endotoxemia.

High-density lipoproteins (HDL) can bind and neutralize lipopolysaccharides (LPS) in vitro and in vivo. HDL can also affect fibrinolytic activity and can directly influence platelet function by reducing platelet aggregation. In this study, the effects of reconstituted HDL (rHDL) on LPS-induced coagulation, fibrinolysis and platelet activation in humans were investigated. In a double-blind, randomized, placebo-controlled, cross-over study, eight healthy male volunteers were injected with LPS (4 ng/kg) on two occasions, once in conjunction with rHDL (40 mg/kg, given as a 4 h infusion starting 3.5 h prior to LPS injection), and once in conjunction with placebo. rHDL significantly reduced LPS-induced activation of coagulation (plasma levels of prothrombin fragment F1 + 2) and fibrinolysis (plasma levels of tissue type plasminogen activator antigen, t-PA). No effect was observed on LPS-induced inhibition of the fibrinolytic pathway (PAI-1) or on the transient thrombocytopenia elicited by LPS. Furthermore, rHDL treatment significantly enhanced the inhibition of collagen-stimulated inhibition of platelet aggregation during endotoxemia, but had no such effect on arachidonate-stimulated platelet aggregation. rHDL treatment per se also reduced collagen-induced platelet aggregation. These results indicate that rHDL modifies the procoagulant state associated with endotoxemia.

Biochemical comparison of the T6 antigen and HLA-A,B antigens.

The human thymic differentiation antigen T6, which was found to be associated with beta 2-microglobulin, was compared to the HLA-A,B antigens. Using a heteroantiserum prepared against denatured heavy chains of HLA-A,B antigens, no cross-reactivity with denatured T6 could be detected. The molecular weight of the protein backbone of T6 was found to be 34,000 as compared to 40,000 for the HLA-A,B antigens. Also, not only was the percentage of carbohydrate of T6 (25-35%) different from the HLA-A,B antigens (10%), but lectin binding studies showed that their sugar composition may differ. The two forms of T6, which previously had been found on MOLT-4 cells, appeared to have different levels of glycosylation, but apparently had the same protein backbone. T6, like HLA, has a hydrophobic domain, since it could be labeled with [125I]iodonaphthylazide. We conclude from these studies that T6 may be a class I MHC antigen which is different from the classical HLA-A,B antigens.

Human cutaneous dendritic cells express two glycoproteins T6 and M241 which are biochemically identical to those found on cortical thymocytes.

Monoclonal antibodies anti-T6 and anti-M241 define unique cell populations within different lineages: cortical thymocytes and dendritic cells in the skin. T6 positive cutaneous dendritic cells are located predominantly in the epidermis and belong to the Langerhans/indeterminate lineage, whereas, most of the M241 positive cells are located in the perivascular regions of the dermis. Biochemical analysis of thymocytes and cutaneous dendritic cells was performed in order to determine whether the reactivity of these antibodies with these cell types is due to sharing of antigenic determinants by two unrelated proteins, or whether similar proteins are present on cells of different lineages. Our results indicate that T6 antigens are borne by the same glycoprotein (49K) on cortical thymocytes and Langerhans/indeterminate cells. Similarly, M241 antigens isolated from thymocytes and cutaneous dendritic cells are found on the same glycoprotein (43K).

Inhibition by immunoglobulins of Staphylococcus aureus adherence to fibronectin-coated foreign surfaces.

Recent data suggest that fibronectin may favor Staphylococcus aureus infection by promoting attachment to either injured tissues or implanted foreign bodies. Using a previously described in vitro assay, we show that promotion of S. aureus adherence by surface-bound fibronectin, adsorbed on polymethylmethacrylate (PMMA) coverslips, is antagonized by antistaphylococcal antibodies present in immunoglobulin G (IgG) purified from human plasma. Among the different organisms tested, the protein A-deficient strain Wood 46 of S. aureus was the most strongly inhibited by purified IgG or whole serum dose-dependently. Bacterial adherence was not influenced by preincubating fibronectin-coated PMMA with either purified IgG or whole serum. However, inhibition of bacterial adherence was directly related to the extent of IgG binding to S. aureus Wood 46. When F(ab')2 fragments of purified IgG were tested in the adherence assay, they could also reduce the interaction between S. aureus Wood 46 and fibronectin-coated PMMA. Two other staphylococcal strains were also tested in the adherence inhibition assay: Whereas the protein A-rich strain Cowan I of S. aureus was moderately inhibited by purified IgG or whole serum, S. epidermidis KH 11 was not at all inhibited by IgG which bound poorly to the bacterial cells. This study has demonstrated that bacterial coating by humoral factors, and specifically IgG, may influence significantly subsequent adherence of S. aureus to surface-bound fibronectin.

Anti-D immunoglobulin in Rh(D) negative volunteers: clearance of Rh(D) positive red cells and kinetics of serum anti-D levels.

Properties of a new anti-D immunoglobulin were assessed in Rh(D) negative healthy male adults. Six volunteers received intravenous, and five volunteers intramuscular injections of 200 micrograms anti-D, 48 hours after pre-treatment with 5 mL of Rh(D) positive erythrocytes. Immediately after intravenous administration of anti-D, a rapid decrease of the Rh(D) positive erythroyctes was noted. After intramuscular injection of anti-D, there was a lag phase of 6 hours until the erythrocytes decreased, and the elimination rate was slower. Twenty-four hours after injection of anti-D, the Rh(D) positive erythrocytes were at the detection limit or no longer detectable in all volunteers. After intravenous administration, anti-D serum levels decreased from 45 ng/mL at 2 hours to 29 ng/mL at 24 hours, whereas after intramuscular administration, anti-D became detectable at 4 hours and increased to 11 ng/mL at 24 hours. During subsequent months, anti-D serum levels decreased at similar rates in both groups. After six months, anti-D was not detectable in any of the volunteers. Thus, the new anti-D immunoglobulin induced elimination of the Rh(D) positive erythrocytes and suggested that Rh(D) immunization of the volunteers was prevented.

Production and characterization of a reconstituted high density lipoprotein for therapeutic applications.

A method is described for the large scale preparation of reconstituted high density lipoproteins (rHDL) suitable for therapeutic use. Apolipoprotein A-I (apoA-I was isolated from precipitates obtained by cold ethanol fractionation of human plasma. This process includes several steps for virus removal and virus inactivation, among them pasteurization. Reconstitution of lipoprotein particles was performed by cholate dialysis using soybean phosphatidylcholine as the lipid source. An apoA-I:lipid ratio of 1:150 (mol:mol) was obtained. Redissolved rHDLs were disc-shaped particles resembling nascent HDL, as assessed by electron microscopy. The method was optimized for low content of free apoA-I protein as well as the low concentration of free lipid. The product was stabilized by lyophilization in the presence of sucrose. In vitro studies show potential effects it the prevention of gram-negative septic shock and in the inhibition of atherosclerosis.

The effect of recombinant interferon-gamma on human monocyte-derived macrophages.

The effect of recombinant interferon-gamma (rIFN-gamma) on human macrophage functions was studied, using monocytes which had matured to macrophages within hydrophobic containers. Following exposure to rIFN-gamma, the number of surface-expressed specific IgG-binding sites was increased. This increase was restricted to high-affinity Fc receptors (FcR), however; low-affinity FcR were not increased in number. Exposure to rIFN-gamma led to an enhanced chemiluminescence (CL) signal in the presence of luminol and a variety of respiratory burst stimuli, such as zymosan, phorbol 12-myristate 13-acetate or IgG-sensitized sheep erythrocytes (EA). In contrast, phagocytosis of EA was markedly depressed in rIFN-gamma-treated cells. Both increase in CL response and decrease in phagocytic activity were manifest after 1 day of treatment and were more pronounced after 2 days. While 5 U/ml of rIFN-gamma was an insufficient dose, 50 to 5000 U/ml yielded significant dose-dependent changes in both functional assays. Thus, using rIFN-gamma as a biological response-modifier, FcR expression and FcR-mediated CL can be dissociated from FcR-mediated phagocytosis.

Diffusion of immunoglobulins into rabbit cornea after subconjunctival injection: experimental demonstration and mathematical model.

To determine whether immunoglobulins of the IgG class diffus up to the corneal center after subconjunctival injection, rabbits were injected with fluorescein-isothiocyanate-labeled human IgG. The inoculum diffused from the entire periphery centrepetally towards the corneal center. The progression of the diffusion front slowed down as the distance to the limbus increased. The first increase of fluorescence in the corneal center was observed on day 6. The intensity increased during the following 10 days despite resorption in the corneal periphery due to the flow of IgG from paracentral toward central areas. The diffusion coefficient of 0.003-0.004 cm2/day was calculated by computer simulation using Fickian diffusion equations adapted for corneal geometry. We conclude that after subconjunctival application, IgG diffuses up to the corneal center with a delay of several days and that the penetration speed decreases as the distance to the limbus increases. This kinetics contributes to our understanding of the role of IgG in corneal pathology and may help to design therapeutic schedules for immunotherapy with IgG.

Modulation of human monocyte Fc receptor function by surface-adsorbed IgG.

Fc receptor-mediated phagocytosis was measured with monocytes subjected to various treatments. Monocytes exposed to IgG during their adherence, or after they had adhered to a surface, experienced functional impairment. This was manifested in the requirement of a higher antibody density on target particle for efficient phagocytosis, and in an enhanced susceptibility to inhibition by fluid-phase IgG. The impairment was found to be due to an interaction of IgG adhering to the surface with the Fc receptors. This effect could be induced with monomeric IgG, devoid of IgG aggregates or immune complexes. IgG coatings that resulted in inefficient Clq fixation promoted considerable functional impairment of monocytes within 1 hr. In addition, the prolonged contact of monocytes with polystyrene in the absence of IgG also led to a functional reduction. The study points to a compromised function of phagocytes exposed to artificial surfaces.

Histidine-rich glycoprotein binding to activated human platelets.

Specific binding of purified histidine rich glycoprotein (HRGP) to human platelets stimulated with either bisdiazoniumbenzidine-crosslinked immunoglobulin G (BDB-IgG), with thrombin or with collagen was dose- and divalent cation dependent. A 5-10-fold increase of platelet bound 125I-HRGP was obtained when 0.5-0.8 x 10(9) platelets/ml were activated with 100 micrograms BDB-IgG/ml, 0.1 U thrombin/ml or 15 micrograms collagen/ml. At maximal binding tested 16,000 molecules of HRGP became bound per platelet, but saturation was not achieved. Such platelet inhibitors as acetylsalicylic acid, prostaglandin E1 and cytochalasin B reduced the capacity of platelets to bind ligand, and by kinetic experiments involving enzymatic digestion of radiolabelled bound HRGP the ligand revealed to remain surface bound rather than being taken up to inner parts of the cell.

Characterisation of a chromatographically produced anti-D immunoglobulin product.

A chromatographic fractionation method has been developed for the production of a liquid-stable anti-D immunoglobulin product for intravenous and intramuscular use. An immunoglobulin fraction, highly enriched with anti-D immunoglobulins, was isolated by cation-exchange column chromatography and further polished, first by anion-exchange chromatography, followed by an aluminium hydroxide gel treatment. The process includes two specific steps for virus inactivation and removal, namely S/D treatment and nanofiltration. The overall anti-D process yield is about 56%. The final product is stabilised with human albumin and glycine and placed in ready-to-use syringes. The anti-D product was shown to be stable in liquid state for at least 30 months at 4 degrees C.

Beta 2-microglobulin from serum associates with MHC class I antigens on the surface of cultured cells.

Beta 2-microglobulin (beta 2-m) is a highly conserved polypeptide (12,000 molecular weight; 12K) noncovalently associated with the heavy chain (45-48K) of the major histocompatibility complex (MHC) class I antigens. Its synthesis is required for expression of the HLA-A/B and H-2K/D heavy chains at the cell surface; beta 2-m is also associated with the human cell-surface antigens T6 and M241 isolated from thymocytes. However, on the T leukaemic cell line MOLT-4 some of the T6 antigens contain a different 12K subunit, termed beta t (refs 3, 7, 8). Purified human beta 2-m can exchange partially both with human beta 2-m associated with HLA-antigens, and with mouse beta 2-m associated with murine alloantigens. As MOLT-4 cells were grown in the presence of fetal calf serum (FCS) and as serum is known to contain some free beta 2-m, we examined whether beta t was bovine beta 2-m which had replaced endogenous beta 2-m on the surface of the cell. Here we show both that beta 2-m from FCS or human serum (HuS) used in cell culture can exchange with beta 2-m on the cell surface, and that beta t is in fact bovine beta 2-m.

Localization of individual lysine-binding regions in human plasminogen and investigations on their complex-forming properties.

After partial digestion of human plasminogen with elastase, followed by chymotryptic cleavage of one of the fragments produced, two polypeptides with molecular weights of approximately 10 000 and with lysine-binding sites still intact were isolated by means of affinity chromatography and gel filtration. One fragment, which was completely sequenced (88 residues), was identified as the fourth kringle, whereas the other, according to partial sequence analysis represented the first kringle. Equilibrium dialysis against 6-aminohexanoic and yielded for the first kringle one high-affinity binding site (Ka = 60 mM-1) and for the fourth kringle one single low-affinity binding site (Ka = 28 mM-1). Moreover, interactions were detected between the first kringle and the N-terminal CNBr fragment of plasminogen and also fibrin. In these cases an additional lysine-binding site, though of low affinity, appears to be involved. Thus, the first kringle seems to play important roles, structurally by contributing to the maintenance of a compact structure of plasminogen through an intramolecular interaction with its N-terminal polypeptide region, and functionally by increasing the fibrin affinity of Lys-plasminogen (plasminogen lacking the first 76 residues) and plasmin.

The thymic differentiation markers T6 and M241 are two unusual MHC class I antigens.

The human beta 2-microglobulin (beta-2m)-associated human thymocyte differentiation antigens T6 and M241 were compared using biochemical techniques. T6 and M241 antigens reside on different molecules with apparent m.w. of 49,000 and 43,000, respectively. Here we show that both proteins have a protein backbone m.w. of 33,000. In addition, T6 and M241 have a large portion of their peptides in common. When we compared the protein backbone m.w. of T6 and M241 with the murine beta-2m-associated thymus leukemia (TL) antigens, we found a considerable difference in size, suggesting that T6 and M241 may not be human homologues of TL antigens and constitute a novel type of major histocompatibility (MHC) class I antigens.