Membrane-bound hemagglutinin mediates antibody and complement-dependent lysis of influenza virus-treated human platelets in autologous serum.

Influenza A virus-treated human platelets were lyzed in autologous serum. Lysis required the presence of antibody and occurred predominantly through activation of the classical complement pathway. Binding of the virus followed by its elution at 37 degrees C resulted in a dose-dependent desialation of the cells with a maximal release of 45% of total platelet sialic acid. In contrast, platelets that had been treated with Vibrio cholerae neuraminidase and from which 55% of total sialic acid had been removed were not lyzed in autologous serum and did not bind C3 as shown in binding assays using radiolabeled monoclonal anti-C3 antibody. Thus, the immune-mediated lysis of virus-treated platelets in autologous serum did not involve neoantigens expressed by desialated cells. To assess the effect of viruses on the platelet surface, treated platelets were incubated with galactose oxidase and sodium [3H]borohydride prior to separation and analysis of the labeled glycoproteins by SDS-PAGE. Viral treatment resulted in a desialation of each of the surface glycoproteins. At the same time, a labeled component of Mr 72,000 (nonreduced) and Mr 55,000 (reduced) was observed that was not present when V. cholerae-desialated platelets were examined in the same way. Immunoblotting experiments performed using antiwhole virus and anti-hemagglutinin antibodies demonstrated this component to be viral hemagglutinin. Involvement of membrane-bound hemagglutinin in antibody and in complement-mediated lysis of virus-treated platelets in autologous serum was supported by the increased lytic activity of a postvaccinal serum containing an elevated titer of complement fixing anti-hemagglutinin antibodies. Binding of a viral protein to the platelet surface provides a model for immune thrombocytopenias occurring during acute viral infections at the time of the specific immune response.

Lysosomal and cytosolic sialidases in rabbit alveolar macrophages: demonstration of increased lysosomal activity after in vivo activation with bacillus Calmette-Guerin.

Sialidase activity was assayed in homogenized rabbit alveolar macrophages using a fluorogenic substrate: sodium 4-methylumbelliferyl-alpha-D-neuraminate. After differential centrifugation one acid-active enzyme (optimum pH 4.2) was detected in the 16,000 X g pellet that contained lysosomes, mitochondria and peroxisomes. A second activity, with an optimum pH of 5.4, was found in the cytosolic fraction. The acid-active sialidase accounted for more than 95% of the total sialidase activity in crude homogenate. When alveolar macrophages were collected from rabbits stimulated with bacillus Calmette-Guerin (BCG), the acid-active sialidase specific activity was increased 2.5-fold whereas other lysosomal enzymes such as N-acetylglucosaminidase and beta-galactosidase were stable. The cytosolic sialidase activity did not change.

Characterization of human pericardial macrophages.

This paper deals with the study of the cell population in 13 samples of normal human pericardial fluid. Large mononuclear cells (LMC) constituted 74.1 +/- 18.5% of the total cell population. These LMC possess the characteristics of macrophages firm adherence to glass intracytoplasmic presence of vimentin without keratin, ultrastructural observation of a lysosomal apparatus cytoenzymatic activities: acid phosphatases, naphthol AS.D acetate esterase and peroxidases, and phagocytosis of Baker's yeasts. All these data clearly show that macrophages are the main component of the pericardial fluid cell population and can be of great significance in the defense mechanisms and physiology of the pericardial space.

Differences in carbohydrate specificities and complement-activating capacity of guinea pig and human antibodies to neuraminidase-treated autologous erythrocytes.

Guinea pig erythrocytes desialated by treatment with neuraminidase from Vibrio cholerae were lyzed in autologous serum through a natural-antibody-dependent activation of the classical complement pathway. Lysis was inhibited when a mannose, glucose, galactose or N-acetyl-glucosamine was added to the incubation mixture. Methyl-alpha- or -beta-D-galactopyranosides were poorly effective and N-acetyl-D-galactosamine was not effective at all. Inhibition of lysis by the carbohydrates was due neither to an anti-complementary effect nor to a modification of the osmotic pressure since: (a) they did not alter the total complement haemolytic activity of guinea pig serum, and (b) they did not inhibit lysis of desialated guinea pig erythrocytes in human serum through activation of the alternative complement pathway. The presence of mannose, glucose, galactose or N-acetyl-glucosamine in the incubation mixture resulted in an impaired fixation of natural auto-antibodies on antigenic sites, namely the T-antigen (Thomsen-Friedenreich), which were unmasked following membrane sialic acid removal. When tested under the same conditions, only small percentage of the normal human population showed the phenomenon of lysis of desialated erythrocytes in autologous serum. Lysis was not due to a particular susceptibility of erythrocytes from these individuals to complement-mediated lysis but to the presence in their serum of complement-activating anti-T antibodies. As expected, the activity of human anti-T antibodies was inhibited by galactose and N-acetyl-galactosamine, which are the immunodominant sugars of the human T-antigen. Mannose and glucose had no effect, and methyl- alpha- or - beta-D-galactopyranosides were almost as effective as galactose. The heterogeneity of the human population with regard to the complement-activating capacity of anti-T antibodies could be of significance for the individual response of the host to an infection by a neuraminidase-producing microorganism. That the immunodominant sugars of the T-antigen were different between humans and guinea pigs was further assessed by absorption experiments. We have demonstrated that guinea pig anti-T antibodies were not removed during contact with desialated human red cells which do not have the mannose specificity, whereas human antibodies were almost entirely retained on desialated guinea pig red cells which, beside mannose, express galactose. These results also suggest that guinea pig antibodies are mostly directed towards mannose and glucose.

A microplate immunoenzyme assay for anti-influenza antibodies.

An enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase is described for the detection and quantitation of anti-influenza virus antibodies. Compared with complement fixation and hemagglutination inhibition tests, ELISA is far superior with respect to sensitivity and reliability. Non-specific viral inhibitors present in sera do not affect the titer in ELISA. Its sensitivity, close to that of radioimmunoassay permits detection of small amounts of antibodies in pulmonary secretions and supernatants from in vitro spleen cell cultures.

Measurement of anti-influenza neuraminidase antibody using a peroxidase-linked lectin and microtitre plates coated with natural substrates.

Neuraminidase-induced removal of sialic acid from natural substrates (desialylation) unmasks saccharides that are specifically recognized by the lectin peanut agglutinin (PNA). We demonstrate that, when a neuraminidase substrate is coated on to the wells of a microplate, it is possible to quantitate the binding of PNA to the desialylated substrate using a peroxidase-conjugated PNA (Po-PNA). The amount of bound PNA correlated directly with the amount of sialic acid removed from the substrate and therefore with the neuraminidase activity. By reacting with specific epitopes that are located near to the enzyme active site, anti-neuraminidase antibodies are capable of inhibiting the virus-induced desialylation of the substrate. Such antibodies therefore reduce the binding of Po-PNA. The advantage of this assay is that since different natural substrates for neuraminidase (erythrocytes, fetuin or gangliosides) can be used to coat the microplates, the capacity of anti-neuraminidase antibody to inhibit the neuraminidase activity towards different types of sialoglycoconjugates can be evaluated. Anti-hemagglutinin or non-specific anti-neuraminidase antibody have no interfering reactivity.

Anticarbohydrate autoantibodies to sialidase-treated erythrocytes and thymocytes in serum from patients with pulmonary sarcoidosis.

PURPOSE: The presence of immunoglobulins and complement in sarcoid granulomata and bronchoalveolar lavage from patients with sarcoidosis suggests that humoral mechanisms may be of importance in granuloma formation. To test this hypothesis, we examined the possibility that antibodies to specific tissue carbohydrates causing alterations and/or dysfunction of immunocompetent cells might be present during sarcoidosis. Because we had previously shown the presence of sialidase activity in bronchoalveolar lavage from these patients, we have looked for the presence of antibodies that recognize sialidase-treated erythrocytes (mostly antigalactose) in the serum of patients with sarcoidosis. Since thymocytes are spontaneously recognized by peanut agglutinin, a lectin that binds galactose, the reactivity of serum from sarcoidosis patients with normal or neuraminidase-treated thymocytes has also been studied. PATIENTS AND METHODS: Serum samples were obtained from the venous blood of patients with biopsy-proven sarcoidosis, most of whom had no extrathoracic symptoms. The mean patient age was 31 years, with a range from 21 to 57 years. There were 12 women and 19 men, and 10% of the patients were smokers. Sarcoidosis was classified as recent if symptoms had been present for less than 1 year and chronic if symptoms had been present for longer than this. Control serum samples were obtained from patients with idiopathic pulmonary fibrosis (n = 9) and from healthy volunteers (n = 15). Furthermore, serum from patients who had previously had sarcoidosis but in whom cures had been achieved was also studied (n = 6). RESULTS: Sialidase-treated erythrocytes were lysed in autologous serum upon incubation at 37 degrees C providing that the serum came from a patient with active disease. Serum from either normal volunteers or patients with resolved sarcoidosis had no significant cytotoxic activity. Lysis proceeded through activation of the classical complement pathway following fixation of autoantibodies. These antibodies were predominantly of the IgM class. They were able to agglutinate neuraminidase-treated thymocytes, whereas untreated thymocytes did not fix the antibodies. Carbohydrate inhibition experiments demonstrated that these antibodies are mostly galactose specific. As this sugar is located immediately below the sialic acid residues in the carbohydrate moiety of membrane glycoconjugates, it is unmasked following sialidase treatment. CONCLUSION: Since galactose has been shown to be present on the membrane of certain subsets of immunocompetent cells (e.g., lymphocytes and macrophages either spontaneously or after stimulation), it is possible that antigalactose antibodies may affect the metabolism of these cells, leading to some of the immune dysfunctions that are observed during sarcoidosis.

Determination of the complement component C2 by ELISA in human serum and bronchoalveolar lavage fluids.

In order to measure the concentration of the human complement component C2 in various biological fluids, an enzyme linked immunosorbent assay (ELISA) was developed. This assay was highly sensitive and allowed to detect as few as 400 pg of C2 in a sample volume of 150 microliters (i.e. 2.6 ng/ml). This is a 10- to 15-fold increase in sensitivity with regard to the conventional hemolytic test. As assessed by an immunoblot analysis, our anti-C2 antiserum was able to detect native C2 as well as the cleavage fragments C2a and C2b generated upon complement activation through the classical pathway. Thus, complement activation involving the classical pathway can easily be evidenced by comparing functional (hemolytic) and immunochemical (ELISA) C2 assays which respectively do not and do reveal activated C2. When C2 was assayed in either normal human serum or bronchoalveolar fluids, in both ELISA and hemolytic tests, a highly significant correlation was observed between the two assays (P less than or equal to 0.01). The specific C2 activity (i.e. functional hemolytic activity/ng C2 assayed in ELISA) was higher in serum than in bronchoalveolar lavage fluids from both normal volunteers and patients with pulmonary diseases.

Modifications of sialidase activity during the monocyte-macrophage differentiation in vitro.

Human mononuclear cells were isolated from peripheral blood by centrifugation over Ficoll Hypaque, followed by adherence to plastic dishes. Monocyte-derived macrophages were obtained after culture for 3 or 5 days of the adherent cells in RPMI medium containing 20% heat-inactivated foetal calf serum. The sialidase activities were assayed in the whole homogenate using sodium 4-methyl-umbelliferyl-alpha-D-neuraminate as substrate, at various pHs, ranging from 3.6 to 6. The in vitro differentiation of monocytes into macrophages from day 0 up to day 5 was accompanied by a significant (P less than or equal to 0.01) increase in the sialidase activity on both a per-cell (+360%) and a per-mg protein in the homogenate (+125%) basis.

Binding of serum autoantibodies to sialidase-treated tracheal epithelial cells. Determination of autoantibodies isotypes in normal and influenza virus infected guinea pig sera.

Cultured epithelial cells isolated from guinea pig trachea were treated with Vibrio cholerae sialidase. The treatment was not cytotoxic and resulted in membrane desialylation as assessed by measurement of sialic acids released, along with an increased fixation of the galactose-specific lectin peanut agglutinin. After incubation in serum from normal guinea pigs, membrane-bound immunoglobulins were detected using peroxidase-labelled antibodies. Sialidase-treated cells bound significantly more IgM than controls (P < 0.0005), whereas binding of IgG was not significantly different between treated and untreated cells (0.1 < P < 0.375); IgA were never detected. In influenza-infected guinea-pigs, as assessed by reactivity with peanut agglutinin, the tracheal and lung epithelium, as well as alveolar cells were hyposialylated. In these animals, the level of serum IgG autoantibodies capable to bind sialidase treated cultured cells increased, while the level of IgM autoantibodies did not change. These autoantibodies may participate in cellular dysfunctions and modified bronchoreactivity that occur during infection of the respiratory tract by sialidase-producing microorganisms, either through activation of the complement system, or subsequently to their reaction with cells expressing membrane complement and/or Fc receptors.

Decreased sialidase activity in alveolar macrophages of guinea pigs exposed to coal mine dust.

The origin of immune dysfunctions that are observed in pneumoconiotic miners still remains unknown. There is evidence that the carbohydrate moiety of membrane glycoconjugates is of primary importance in many functions of immunocompetent cells. The glycosylation, and especially the sialylation level of membrane components of various lymphocyte and macrophage subsets, vary depending on the state of cellular differentiation and activation. Sialidases, which may regulate the amount of sialic acids exposed on the cell membrane, can thus be considered as immunoregulatory enzymes. In this report, the sialidase activity has been measured in alveolar macrophages (AM) and in cell-free bronchoalveolar lavage fluid (BALF) from guinea pigs exposed for 4 months to coal mine dust at a concentration of 300 mg/m3. The samples were collected by bronchoalveolar lavage 2 months after cessation of exposure. The sialidase activity in the cell-free fluid and in the purified alveolar macrophages showed a 10-fold decrease (p less than 0.001). Kinetic parameters of the enzyme such as Km and optimum pH did not change. This changed activity was specific for sialidase, as two other lysosomal glycosidases, beta-galactosidase and N-acetylglucosaminidase, showed unchanged activities. These results suggest the possibility that, by inducing a decreased sialidase activity, exposure to coal mine dust may lead to a modified expression of AM membrane-associated sialic acids giving rise to altered immune functions (i. e., phagocytosis, antigen processing, response to cytokines, etc.).

An enzyme-linked lectin assay for sialidase.

A procedure for the detection of low activities of sialidase (= neuraminidase) is described. Natural substrates for sialidase (human erythrocytes, fetuin or gangliosides) were coated onto the wells of microplates and incubated at 37 degrees C with the enzyme. Sialidase-induced desialylation of these natural substrates unmasks saccharides that are specifically recognized by the peanut agglutinin lectin (PNA). The use of a peroxidase-conjugated PNA (Po-PNA) allowed the binding of the lectin to the desialylated substrate to be quantified. The amount of bound Po-PNA correlated directly with the amount of sialic acid released from the substrate, and therefore with the sialidase activity. With this method, it was possible to detect sialidase activity associated with bacteria, myxoviruses and cells from higher organisms. This method may have important clinical implications as the use of ELISA allows automation and concurrent analysis of numerous samples.

Activation of the hsp70 promoter by environmental inorganic and organic chemicals: relationships with cytotoxicity and lipophilicity.

Stress proteins (heat shock proteins, HSPs) have been proposed as general markers of cellular aggression and their use for environmental monitoring is often suggested. The aim of this work was to study the potency of various environmentally relevant organic and inorganic chemicals to induce the expression of the HSP70 marker. For this purpose, we used an established HeLa cell line containing the chloramphenicol acetyl transferase (CAT) gene under the control of the hsp70 promoter. The screening of three metallic and 15 organic chemicals revealed differences in their capacities to induce the hsp70 promoter. The three metals tested (cadmium, zinc and mercury) were able to induce a stress response. Some organochlorine compounds (chlorophenol derivatives, tetrachlorohydroquinone, 3, 4-dichloroaniline, ethyl parathion and 1-chloro-2,4-dinitrobenzene) induced a response, whereas other common halogenated pesticides or aromatic hydrocarbons (e.g. benzo(a)pyrene, 2, 4-dichlorophenoxyacetic acid, endosulfan, diuron, 4-nonylphenol) did not. The potency to induce hsp70 was significantly correlated to the octanol-water partition coefficient (log K(ow)) of the inducing chemicals, except for 1-chloro-2,4-dinitrobenzene and ethyl parathion. Cytotoxicity assays run in parallel to the induction measurements revealed that the three metals were effective at non cytotoxic doses whereas all organic compounds, except tetrachlorohydroquinone and 1-chloro-2,4-dinitrobenzene, induced the promoter at cytotoxic doses. These results suggest that hsp70 is induced by different mechanisms of toxicity. We propose that this model can be used in mechanistic studies for the detection of toxic effects of certain pollutants.

Early cellular and biochemical alveolar responses following intra-tracheal inoculation with low dose of asbestos and quartz.

The study of broncho-alveolar lavage harvested from rats intratracheally dusted with chrysotile (0.5 mg), leached chrysotile (0.5 mg), crocidolite (0.5 mg) and quartz (0.5 and 5 mg) indicated: 1. A cytotoxic lysis of alveolar macrophages in relation to phagocytosis of dust particles in the following decreasing order: quartz, crocidolite, chrysotile. 2. Regarding cell intensity and duration while asbestos, even leached chrysotile, gave merely an early and transient response. This cell recruitment concerned mostly PMN leukocytes and at a less extent alveolar macrophage. 3. Cell recruitment was associated with an increased protein and phospholipids alveolar content. The increase of proteins came probably mostly from an inflammatory serum exudation. However, an increase synthesis of complement C3 and phospholipids is not excluded.

Induction of the hsp70 Gene Promoter by Various Anticancer Drugs.

HeLa cells containing the chloramphenicol acetyl transferase (CAT) gene under the control of the hsp70 promoter have been exposed in vitro to various anticancer drugs. Cisplatin induced CAT production with a dose-effect relationship at a non-cytotoxic dose, whereas no induction was detected with carboplatin. Etoposid induced a significant response at a cytotoxic concentration. The limited positive response with doxorubicin, daunomycin and mitoxantrone was not statistically significant. These chemicals are known to produce reactive oxygen species and induce apoptosis. No induction of the hsp70 promoter could be detected with the other cytostatic compounds that have been tested such as base analogues (5-fluorouracil, cytosine arabinoside 3'-MP), inhibitors of DNA synthesis (amethopterin, aminopterin), antimitotics (vinblastine, colchicine), and alkylating (streptozotocine, carboplatin, melphalan) or intercalating agents (bleomycin). In addition, the role of the transcription inhibitory activity of doxorubicin in this model is evidenced and the consequent question of the suitability of the reporter gene system is discussed. Our results suggest that specific genotoxic compounds are not able to induce the hsp70 promoter, and are in agreement with the concept that stimulation of HSP70 synthesis occurs through a biochemical process involving proteotoxicity.

Use of transepithelial electrical resistance in the study of pentachlorophenol toxicity.

The toxicity of pentachlorophenol (PCP), a polluting substance believed to exert a narcotic effect, was assayed using the Caco-2 cell line as a model. In order to assess this toxicity as fully as possible, several viability tests, each examining different endpoints, have been used. Neutral red uptake was found to be more sensitive to PCP than MTT and Alamar Blue tests. Transepithelial electrical resistance (TEER) was shown to be the most sensitive to PCP at concentrations and exposure times where the Alamar Blue, LDH leakage and Blue Dextran passage did not evidence any effect. Blue Dextran passage and optical microscopy revealed cellular detachment at concentrations where LDH and Alamar Blue showed little or no cytotoxicity. Thus, PCP seems to affect the integrity of the intestinal barrier at levels where no cytotoxicity is seen. Our results support the notion that TEER can be used as a very sensitive method for evaluating membrane-perturbing toxicants.

Use of the caco-2 model in the screening of polluting substance toxicity.

The aim of this work was to investigate the oral toxicity of representative chemicals chosen from each class of the list of 132 substances present in industrial effluents after the EEC Directive 76-464. Owing to its characterization as a model of the intestinal epithelium, the CaCo-2 cell line model was chosen. Cytotoxicity was assayed using the tetrazolium blue (MTT) test. For most of the substances, a linear correlation was observed between the octanol/water partition coefficient (log Kw) and the median inhibition concentration (IC(50)). This relationship between lipophilicity and toxicity is the hallmark of a narcotic mechanism of action. However, diethylamine appeared more toxic than the correlation would predict. Other amines were then tested (tert-butylamine, n-butylamine and benzylamine). All of these did not fit into the baseline correlation. The IC(50) were corrected by taking into account only the non-ionized, lipid insoluble, concentration at pH7.3. The amines still did not fit into the correlation, reinforcing the idea of a non-narcotic mechanism. The toxicity of a large number of substances can thus be predicted from their physico-chemical properties only when the substances exert a direct and non-specific effect. The amines appeared more toxic than substances with the same partition coefficient, showing that knowledge of the only lipophilicity is too restrictive to predict toxicity.

Identification of immunotoxic effects of chemicals and assessment of their relevance to man.

Immunotoxicity is defined as the adverse effects of foreign substances (xenobiotics) on the immune system. Two types of effects are possible: immunosuppression (which may result in an increased susceptibility to infection or to the development of tumours) and immunopotentiation (which may manifest as an allergy or as autoimmunity). There is, as yet, little evidence that well controlled occupational exposure to industrial chemicals has led to clinically significant immunosuppression. In contrast, a number of industrial chemicals have been shown to cause immunopotentiation in exposed populations, producing occupational asthma and contact dermatitis and possibly autoimmunity. In experimental models, immunosuppression (usually assessed by in vivo or in vitro immune function tests) has been induced by a wide range of chemicals but there are a few reports of the immunosuppression leading directly to an increased susceptibility to infection or to the development of tumours. Predictive experimental models are available for type IV allergic reactions, but the identification of chemicals that have a potential to cause other types of allergy or autoimmune reactions requires further research and the development and validation of new animal models. It is considered that routine subacute and chronic toxicity studies should include a full gross and histopathological assessment of the lymphoid organs to more accurately detect the potential of a chemical to cause immunotoxicity. Should such studies indicate that a substance has affected the immune system directly, an assessment of overall immune competence and function tests may be necessary using dose levels below those which cause frank toxicity. However, precise interpretation of immune function tests in terms of their relevance to human health requires an improved understanding of the extent of the functional reserve of the immune system. A strategy for assessing immunotoxicity in exposed human populations demonstrates a need for reliable clinical assessment, accurate medical record-keeping, an environmental and biological monitoring for levels of contaminating chemicals and the judicious use of well-validated immune function tests.

Lung alterations in guinea-pigs infected with influenza virus.

Guinea-pigs were infected intranasally with influenza A Hong Kong 68 (H3N2) virus. Infective particles were re-isolated from lung homogenates up to 3 days after inoculation and indicated local replication. The subsequent lung inflammatory stages were studied by light microscopy, scanning and transmission electron microscopy (TEM). Lung alterations appeared after 24 h and intensified up to 7 days after virus inoculation, progressively decreasing until 3 weeks thereafter. The damage was reversible and complete restoration of structure was obtained within 5 weeks. The lesions commenced with the infiltration of bronchiolar and alveolar walls by polymorphonuclear cells, histiocytes and macrophages. A purulent exudate was seen to occupy the bronchiolar lumen. Cilia disappeared from tracheal and bronchiolar epithelia. Tracheal epithelium desquamated in some animals. TEM examination showed deterioration in type I pneumocytes, an increase in type II pneumocytes and concomitant damage to alveolar capillaries. Alveolar oedema and fibrinous deposits were seen. The pleura presented slight modifications. These results show that infection of guinea-pigs with influenza virus is a useful model for the study of lung pathology associated with a non-lethal respiratory viral infection.

[Ozone and the immune system]. / Ozone et système immunitaire.

This review focuses on the effect on health of changes in the immune system secondary to ozone exposure and on various mechanistic hypotheses put forward. Beyond the problems related to the variability of study criteria (e.g. age, sex, concentration and duration of different types of exposure, the slightly volatile nature of ozone and the complexity of the immune system), ozone may induce immunostimulation as shown by intensified allergic phenomena or immunosuppression expressed by increased sensitivity to bacterial infections. Different functions of the immune response (for example macrophage and polynuclear phagocytic and bactericidal activity, NK activity, cytokine and antibody production ...) are affected. In terms of risk, the consequences of these changes depend on their intensity, their perennial nature and their association with particular genetic characteristics or other forms of external aggression, for example infection. The effect of exposure to a mixture of pollutants with unknown interactions should also be taken into consideration. Finally, the problem of normal but possibly exaggerated immune response to a compound whose allergenicity may have been modified by ozone must also be taken into account.