Molecular characteristics of cytochrome b558 isolated from human granulocytes, monocytes and HL60 and U937 cells differentiated into monocyte/macrophages.

Enriched cytochrome b558 preparations were obtained from human mature monocytes (MN) and retinoic acid plus interferon gamma induced human myeloid leukemia cell lines HL-60 and U937, using an adaptation of the procedure described by A.W. Segal (Nature (1987) 326, 88-91) for purification of cytochrome b558 from human polymorphonuclear leukocytes (PMN). Spectral characteristics of cytochrome b558 were determined and found to be independent of cell type and specific heme b content of the preparation. To increase the sensitivity of the spectral assay, analysis in the gamma band were used and delta epsilon 427-413 was determined to be equal to 158 mM-1 cm-1. An alpha beta type heterodimeric cytochrome b558 was found for PMN and MN by the concordant elution of heme b spectral activity from heparin agarose and the detection of two polypeptide chains by SDS-PAGE. The expression of the lighter polypeptide alpha chain in the various human monocyte-like cell lines was assessed and its identity, as a component of cytochrome b, was confirmed by immunodetection using a rabbit polyclonal antibody reacting with the alpha subunit of PMN cytochrome b558. Immunoblotting studies detected the alpha subunit in monocyto-macrophagic differentiated HL-60 and U 937 cells and mature MN at 22 kDa, but not in uninduced cells which did not express the respiratory burst. Whatever the specific content or the cell origin of the cytochrome b558-enriched preparations, the heme b binding site was shown to be associated with the alpha subunit defined by a constant molecular mass of 22 kDa, as evidenced by the finding of a constant ratio between the silver stained band intensity and the corresponding heme b amount. The heavy polypeptide beta chain from MN cytochrome b was found to have a significantly higher molecular weight than the beta subunit from PMN at 94 +/- 5 kDa instead of 90 +/- 4 kDa. In contrast, in cytochrome b preparations from induced monocyto-macrophagic cells, isolated with a low heme specific content, the variability in the detection of the staining intensity of the beta band either in SDS-PAGE or immunodetection reactivities precludes accurate definition of its molecular mass and estimation of the stoichiometry between the alpha and beta subunits in the differentiated cells. However, wheat-germ agglutinin binding studies indicated the presence of N-glycosylated protein in the range of 85-110 kDa.

Pholasin: a new chemiluminescent probe for the detection of chloramines derived from human phagocytes.

We have previously reported that normal human polymorphonuclear neutrophils (PMN) release taurine-chloramine, a long-lived oxidant, in response to stimulation by phorbol myristate acetate (PMA) or opsonized zymosan in the presence of exogenous taurine. We now describe a new, simple, and highly sensitive method for the detection of chloramines, including taurine-chloramine, using the chemiluminescent probe Pholasin, the luciferin of the mollusc Pholas dactylus. Taurine-chloramine (N-chlorotaurine) detection was assessed with both a colorimetric method (based on the oxidation of potassium iodide) and with the Pholasin-dependent chemiluminescence (CL) method. The taurine-chloramine concentration in PMN supernatants determined using the potassium iodide (KI) method correlated closely with Pholasin-dependent CL. This CL was also assessed in nonoxidative conditions. No taurine-chloramine was detected in supernatants of lymphocytes and PMN from patients with an oxidative burst defect (chronic granulomatous disease, CGD) with the KI method, but Pholasin-dependent CL was consistently observed. The use of methionine, a specific chloramine scavenger in our incubation conditions, allowed us to define a methionine-inhibitable fraction of Pholasin-dependent CL (i.e., chloramine-induced CL).

Characterization of a recombinant proteinase 3, the autoantigen in Wegener's granulomatosis and its reactivity with anti-neutrophil cytoplasmic autoantibodies.

Using the baculovirus/insect cells system, we have expressed a recombinant proteinase 3 (PR3) -- the neutrophil-derived serine protease autoantigen in Wegener's granulomatosis -- as a glycosylated intracellular and membrane-associated protein. Oligosaccharides accounted for the difference in molecular weights between recombinant (34 kDa) and neutrophil-PR3 (29 kDa). Whereas rabbit-anti-PR3 IgG recognized both recombinant and neutrophil-derived PR3, autoantibodies from Wegener patient sera recognized only neutrophil-derived PR3. Although oligosaccharides were not involved in PR3 epitope recognition, autoantibodies did not recognize the amino acid primary structure of recombinant PR3. Improper disulfide bond formation and/or lack of post-translational events in insect cells, may affect the conformation and/or lack of post-translational events in insect cells, may affect the conformation of PR3, precluding its reactivity with sera from WG patients.

Glutathione antioxidant system as a marker of oxidative stress in chronic renal failure.

A profound imbalance between oxidants and antioxidants has been suggested in uremic patients on maintenance hemodialysis. However, the respective influence of uremia and dialysis procedure has not been evaluated. Circulating levels of copper-zinc superoxide dismutase (CuZn SOD), glutathione peroxidase (GSH-Px), and reductase (GSSG-Rd), total GSH and GSSG were determined in a large cohort of 233 uremic patients including 185 undialyzed patients with mild to severe chronic renal failure, and 48 patients treated by peritoneal dialysis or hemodialysis. Compared to controls, erythrocyte GSH-Px and GSSG-Rd activities were significantly increased at the mild stage of chronic uremia (p < .001), whereas erythrocyte CuZn SOD activity was unchanged, total level of GSH and plasma GSH-Px activity were significantly decreased, and GSSG level and GSSG-Rd activity were unchanged. Positive Spearman rank correlations were observed between creatinine clearance and plasma levels of GSH-Px (r = .65, p < .001), selenium (r = .47, p < .001), and GSH (r = .41, p < .001). Alterations in antioxidant systems gradually increased with the degree of renal failure, further rose in patients on peritoneal dialysis and culminated in hemodialysis patients in whom an almost complete abolishment of GSH-Px activity was observed. In conclusion, such disturbances in antioxidant systems that occur from the early stage of chronic uremia and are exacerbated by dialysis provide additional evidence for a resulting oxidative stress that could contribute to the development of accelerated atherosclerosis and other long-term complications in uremic patients.

Chemiluminescence monitoring of phagocyte oxidative metabolism in mice bearing polyacrylamide induced granulomas.

A technical protocol was recently described by Fauve et al. (J. Immunol. Methods 1983, 64, 345) for inducing subcutaneous granuloma with polyacrylamide microbeads. The present study using this technique demonstrates that the capacity of host phagocytes to generate reactive oxygen species can be easily monitored by chemiluminescence, both locally in granuloma infiltrating cells and at sites remote from the inflammatory reaction, i.e., within microamounts of whole blood and in spleen cells. We observed that both resting and stimulated (zymosan or phorbol-myristate acetate) production by C57BL/6 mouse phagocytes are significantly higher in granulomas induced with high porosity polyacrylamide beads (P300) than in those induced with beads of low polyacrylamide porosity (P4). Since this selective modulation of phagocyte oxidative metabolism is also detectable within microamounts of whole blood and in spleen cells, it could serve as a model for investigating the role of reactive oxygen species in the inflammatory reaction.

Benzodiazepine anesthesia in humans modulates the interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6 responses of blood monocytes.

The influence of sedative and anxiolytic benzodiazepines on human monocyte function was assessed in 11 patients undergoing anesthesia prior to control endoscopy of the urinary tract. A single i.v. injection of 0.08 mg/kg midazolam induced a marked and delayed inhibition of the lipopolysaccharide-induced production of interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6 by monocytes isolated from peripheral blood. Corticosteroids were not responsible for the observed immunosuppression. These studies demonstrate that, when administered in man, benzodiazepines markedly alter the capacity of monocytes to synthetize major mediators of the host inflammatory response.

In vivo soluble tumor necrosis factor receptor release in OKT3-treated patients. Differential regulation of TNF-sR55 and TNF-sR75.

Administration of monoclonal antibodies to CD3 triggers acute and massive release of several cytokines, including tumor necrosis factor alpha (TNF-alpha), essentially T cell-derived. This cytokine release is responsible for the spontaneously reversible acute clinical syndrome observed in most OKT3-treated patients. We found that the first OKT3 injection in human renal allograft recipients led to the release in significant amounts of soluble TNF receptors (TNF-sR55 and TNF-sR75) that are considered main natural inhibitors of TNF bioactivity. As for OKT3-induced TNF-alpha, peak TNF-sR levels were observed 1 hr postinjection, and this release was limited to the first monoclonal antibody injection. A distinct regulation of OKT3-mediated release of TNF-sR75 and TNF-sR55 was observed, since (1) in clear contrast with OKT3-mediated TNF-sR75 induction, TNF-sR55 release was completely blocked by a high dose of corticosteroids prior to OKT3 injection and (2) secretion of TNF-sR75 but not TNF-sR55 correlated with immunoreactive TNF-alpha release. In hemodialyzed patients prior to transplantation and OKT3 treatment, a condition characterized by chronic TNF-alpha release, TNF-sR efficiently block TNF bioactivity. In contrast, the system is overwhelmed by the massive acute TNF-alpha release that follows the first OKT3 injection: in such a condition TNF-sR looses its capacity to counteract TNF bioactivity.

T cells and B cells in chronic renal failure.

Recent knowledge into the pathophysiological mechanisms mediating the immune abnormalities characteristic of end-stage renal disease (ESRD) has focused on the dual activation versus deficiency state of immunocompetent cells. Despite major advances in renal replacement therapy, notably hemodialysis, no significant improvement in the immune status of uremic patients has been achieved. After a brief review of the role of T cells and B cells in the normal immune response, the functional and phenotypic T and B cell abnormalities observed in uremic patients are presented. Special emphasis is placed on our recent findings indicating that these abnormalities are observed at an early stage in the course of chronic renal failure, worsen with the progression of uremia, and are exacerbated by the dialysis procedure. The previous hypotheses that could reconcile the so-called Janus-faced behavior of T cells in uremia are updated in light of the recent findings obtained in the search of therapeutic strategies that could counteract the impaired responsiveness of patients with ESRD to vaccination against hepatitis B virus. Perspectives of research aimed at elucidating the respective role of T helper cell subpopulations (Th1 and Th2) could contribute to understanding of the mechanisms of the multifaceted process of uremia-related immune dysregulation and of the rationale for possible immunointervention strategies.

Deleterious effects of cardiopulmonary bypass. A prospective study of bubble versus membrane oxygenation.

A number of hematologic and immunologic parameters that reflect erythrocyte and platelet damage and host defense mechanisms against infection were studied in 20 patients undergoing cardiopulmonary bypass during coronary operations. The patients were randomly assigned to a group in which a bubble oxygenator or a hollow-fiber membrane oxygenator was used. Hemolysis, thrombocytopenia, and significant release of beta thromboglobulin occurred in patients from the bubble oxygenator group and, to much lesser extent, in patients from the membrane oxygenator group. Polymorphonuclear leukocytes and monocytes from bubble oxygenator patients demonstrated increased generation of reactive oxygen species in the resting state and in the presence of the stimulating agents N-formyl-methionyl-leucyl-phenylalanine, concanavalin A, and opsonized zymosan, as compared with cells from membrane oxygenator patients. No difference was found between bubble and membrane oxygenator patients in the time of occurrence or intensity of leukopenia during bypass, of leukocytosis at the end of bypass, nor in the rate of complement activation, as assessed by quantitation of plasma C3a antigen. Complement activation was dependent on the alternative pathway. Immunoglobulin M concentration significantly decreased during bypass in both groups of patients. The serum opsonizing capacity for endotoxin and serum bactericidal activity for Serratia marcescens were decreased in both groups, mainly because of hemodilution, although they were additionally affected by bubble oxygenation. Several deleterious hematologic consequences of cardiopulmonary bypass can be minimized by the use of a membrane oxygenator. However, complement activation remains a potential risk factor even in membrane oxygenator patients and requires further investigation to obtain better hemocompatible materials for cardiopulmonary bypass circuits.

Importance of oxidatively modified proteins in chronic renal failure.

Considerable evidence has accumulated that chronic uremia is associated with a multifactorial immunoinflammatory syndrome, which occurs early in the course of renal failure, is accentuated with the progression of uremia, and culminates in maintenance dialysis therapy. We previously described the presence of a circulating oxidized plasma protein named advanced oxidation protein products (AOPPs). Beyond evidence that AOPPs represent an exquisite marker of oxidative stress, their role(s) in the pathophysiology of chronic renal failure and dialysis-related complications might be of great importance. Regarding the mechanisms of generation of AOPP, we underscore the importance of the chlorinated oxidants, previously solely considered as microbicidal agents, in the generation of AOPP. Indeed, AOPPs appear to act as true inflammatory mediators since they are able to trigger the oxidative burst in neutrophils as well as in monocytes. Thus, it is hypothesized that the AOPPs, which arise from the reaction between chlorinated oxidants and plasma proteins, constitute a new molecular basis for the deleterious activity of oxidants, and they could be considered to be true mediators of the proinflammatory effect of oxidative stress in uremia.

Role of oxidized low-density lipoprotein in the atherosclerosis of uremia.

Lipoprotein oxidation is involved in the genesis of atherosclerosis. In chronic renal failure (CRF), oxidative stress is enhanced because of an imbalance between pro-oxidant and antioxidant systems. Oxidative modifications of low-density lipoproteins (LDLs) occur not only at the level of lipid moiety, but also of protein moiety. We have shown that oxidation of LDL by hypochlorous acid (HOCl) in vitro, reflecting increased myeloperoxidase activity in vivo, leads to modifications of apoliproteins such that the latter in turn are capable of triggering macrophage nicotinamide adenine dinucleotide phosphate-oxidase activation. These oxidative changes of LDL protein moiety, if shown to occur to a significant extent in uremic patients in vivo, may represent an important alternative pathway in the pathogenesis of atheromatous lesions.

Homocyst(e)ine, oxidative stress, and endothelium function in uremic patients.

Moderate hyperhomocyst(e)inemia and impaired endothelium-dependent vasodilatation are present in uremic patients. However, the precise mechanism(s) underlying the link between moderate hyperhomocyst(e)inemia and endothelium dysfunction in uremic patients remains to be determined. Experimental and clinical evidence have led to the suggestion that moderate hyperhomocyst(e)inemia may predispose to endothelium dysfunction through a mechanism that involves generation of reactive oxygen species and a decrease in nitric oxide bioavailability. Recent preliminary findings in uremic patients provide support for some aspects of this suggestion. These data must be confirmed in additional studies. Moreover, the relative importance of homocysteine-induced oxidant stress versus other potential mechanisms of endothelium dysfunction in these patients remains to be determined.

Activation of phagocyte oxidative metabolism by opsonized Plasmodium falciparum merozoites.

A luminol-dependent chemiluminescence (CL) method was used to measure the oxidative burst of phagocytes triggered by antimalarial antibodies and Plasmodium falciparum merozoites. A specific antibody-dependent increase in chemiluminescence response was obtained using both polymorphonuclear leukocytes and monocytes as effector cells. Using various sera from malaria infected subjects it was observed that antibodies involved in the increased chemiluminescence responses were encountered at higher levels in sera from protected subjects than in those susceptible to clinical manifestations. In the conditions used the assay shows substantial intertest variation but appears of interest to assess the protective status of groups of exposed individuals.

The advanced glycation endproduct pentosidine and monocyte activation in uremia.

RATIONALE: Advanced glycation endproducts (AGEs) contribute to the pathogenesis of vascular complications in diabetes, aging and end-stage renal disease (ESRD). Immune abnormalities in patients with chronic renal failure and those treated by dialysis contribute to high rates of morbidity and mortality. We therefore sought a relationship between a circulating marker of immune dysfunction and plasma levels of the AGE pentosidine. METHOD: We studied non-diabetic patients with mild to advanced renal failure (n = 60), and with ESRD treated by hemodialysis (HD) (n = 44) and peritoneal dialysis (PD) (n = 19). The plasma protein content of the well characterized AGE, pentosidine was measured using HPLC. In the same samples the monocyte activation product neopterin was measured by RIA. RESULTS: Plasma levels of pentosidine and neopterin increased in parallel with the progression of renal failure. Pentosidine and neopterin were highly correlated in all patients even after adjustment for Ccr. This correlation was also present in patients with ESRD. CONCLUSION: These data suggest that the AGE pentosidine is associated with monocyte activation in renal failure, an interaction which may contribute to accelerated rates of complication and death by as yet unknown mechanisms.

Uremic toxicity: present state of the art.

The uremic syndrome is a complex mixture of organ dysfunctions, which is attributed to the retention of a myriad of compounds that under normal condition are excreted by the healthy kidneys (uremic toxins). In the area of identification and characterization of uremic toxins and in the knowledge of their pathophysiologic importance, major steps forward have been made during recent years. The present article is a review of several of these steps, especially in the area of information about the compounds that could play a role in the development of cardiovascular complications. It is written by those members of the Uremic Toxins Group, which has been created by the European Society for Artificial Organs (ESAO). Each of the 16 authors has written a state of the art in his/her major area of interest.

[Dysregulation of the immune system in chronic uremic and hemodialysed patients]. / Dysrégulation du système immunitaire chez l'urémique chronique et le dialysé.

Concomitant immune deficiency and activation of immuno-competent cells, together with a disequilibrium between inflammation-inducing cytokines and their specific natural inhibitors is the basis of our current understanding of immune system dysregulation in patients with chronic uraemia. These anomalies may even be accentuated by dialysis. Clinically, bacterial infections, viral hepatitis and amyloidosis all play important roles. Humoral factors include abnormal immunoglobulin response to specific antibodies and complement activation. The response of T lymphocytes, long sought as the origin of the immunodeficiency associated with chronic uraemia, is also significantly decreased in these patients. The decreased antibody responses to specific stimuli may be related to B cell dysfunction. Monocyte and polymorphonuclear cell reactions are also perturbed. A deficiency in natural killer cells is observed although the mechanisms involved and the consequences are still debated. The factors determining the anomalies leading to immune system dysregulation in chronic uraemia and dialysis and their relationship with the reduction in active nephron mass as well as their metabolic and/or endocrine consequences remain to be fully described. A better understanding of the mechanisms involved should lead to new strategies for immuno-intervention in patients with chronic renal failure and help in optimizing haemodialysis.