The effects of neutrophil-generated hypochlorous acid and other hypohalous acids on host and pathogens.

Neutrophils are predominant immune cells that protect the human body against infections by deploying sophisticated antimicrobial strategies including phagocytosis of bacteria and neutrophil extracellular trap (NET) formation. Here, we provide an overview of the mechanisms by which neutrophils kill exogenous pathogens before we focus on one particular weapon in their arsenal: the generation of the oxidizing hypohalous acids HOCl, HOBr and HOSCN during the so-called oxidative burst by the enzyme myeloperoxidase. We look at the effects of these hypohalous acids on biological systems in general and proteins in particular and turn our attention to bacterial strategies to survive HOCl stress. HOCl is a strong inducer of protein aggregation, which bacteria can counteract by chaperone-like holdases that bind unfolding proteins without the need for energy in the form of ATP. These chaperones are activated by HOCl through thiol oxidation (Hsp33) or N-chlorination of basic amino acid side-chains (RidA and CnoX) and contribute to bacterial survival during HOCl stress. However, neutrophil-generated hypohalous acids also affect the host system. Recent studies have shown that plasma proteins act not only as sinks for HOCl, but get actively transformed into modulators of the cellular immune response through N-chlorination. N-chlorinated serum albumin can prevent aggregation of proteins, stimulate immune cells, and act as a pro-survival factor for immune cells in the presence of cytotoxic antigens. Finally, we take a look at the emerging role of HOCl as a potential signaling molecule, particularly its role in neutrophil extracellular trap formation.

Response of Pseudomonas aeruginosa to the Innate Immune System-Derived Oxidants Hypochlorous Acid and Hypothiocyanous Acid.

Pseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host's immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system.

Reactive Oxygen Species and Neutrophil Function.

Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling.

Assessment of IgG avidity against pertussis toxin and filamentous hemagglutinin via an adapted enzyme-linked immunosorbent assay (ELISA) using ammonium thiocyanate.

Antibody avidity, defined as the strength of binding between antibody and antigen, represents a functional measure of affinity maturation of antibodies. Determination of the antibody avidity is usually performed separating high and low avidity antibodies by dissociating agents, but measurement of the antibody avidity in humans is rather complicated, due to the heterogeneity of the antibodies produced in response to complex antigens, e.g. after vaccinations. The purpose of the present study was to evaluate the experimental determinants of the assessment of avidities of IgG antibodies directed against pertussis toxin (IgG-anti-PT) and filamentous hemagglutinin (IgG-anti-FHA) produced after pertussis vaccination using an adapted ELISA and ammonium thiocyanate (NH(4)SCN) as dissociating agent. Our experiments revealed that the results of avidity testing depend very much on experimental conditions and may over- or underestimate the relative avidity of IgG-anti-PT and IgG-anti-FHA antibodies. Whereas in our findings avidity seems to be independent from the initial antibody concentration in a wide range of measures, RAI depends on NH(4)SCN concentration, time of incubation and temperature of the reaction. The presented method allows an accurate measurement of the IgG antibody avidity against both Bordetella pertussis antigens PT and FHA, using NH(4)SCN as chaotropic agent in concentrations lower than 3.0M for 20 min time of incubation at 37 °C. Different experimental conditions in testing pertussis-specific IgG antibody avidity should be considered in interpretation and comparability of data of different studies.

Induction of protective immunity in rabbits by coadministration of inactivated Pasteurella multocida toxin and potassium thiocyanate extract.

Pasteurella multocida is a bacterial pathogen that causes rhinitis (snuffles), pneumonia, otitis media, septicemia, metritis, and death in domestic rabbits. Currently, there are no effective vaccines to prevent infection by this organism. Subcutaneous (s.c.) immunization with either exotoxin or thiocyanate extracts of P. multocida induces partial protection in rabbits. Since disease begins at mucosal sites, induction of local immunity may be important in preventing systemic disease. Little is known concerning the efficacy of intranasal (i.n. ) administration of these antigens in inducing protective mucosal immunity to P. multocida in rabbits. The purpose of this study was twofold: (i) to investigate the effectiveness of vaccination with purified P. multocida toxin (PMT) and a potassium thiocyanate extract of P. multocida (CN) in combination and (ii) to evaluate the efficacy of administration of these antigens i.n. versus s.c. Forty-eight rabbits were randomly divided into eight different treatment groups. Rabbits received either one or both antigens by either s.c. or i.n. administration. Following vaccination, each group received an i.n. challenge of P. multocida. Rabbits vaccinated with both antigens i.n. or s.c. had a 100% survival rate, few or no bacteria in the liver and lungs, high serum immunoglobulin G (IgG) and IgM antibody titers, and significant numbers of IgG antibody-secreting cells (ASC) in the spleen and tracheobronchial lymph node. Rabbits vaccinated i.n. had significant nasal and bronchoalveolar lavage IgA antibody levels. Rabbits vaccinated with only one antigen, either PMT or CN, had lower antibody titers, moderate to severe liver and lung infections, and fewer ASC compared to rabbits receiving both antigens. Rabbits in the control groups had moderate to severe liver and lung infections. This study indicates that i.n. immunization with both PMT and CN induces an effective response against homologous P. multocida challenge.

[The importance of the endogenous agent and environmental factor thiocyanate for nonspecific and specific resistance from the hygienic viewpoint]. / Zur Bedeutung des endogenen Wirkstoffs und Umweltfaktors Thiocyanat für die unspezifische und spezifische Resistenz aus hygienischer Sicht.

Thiocyanate (previous designation rhodanide, SCN-) is a physiological substance which is ubiquitously spread in the animate nature. As an essential constituent of cell it participates in important physiological resp. biochemical processes. From the hygienic and microbiological point of view the occurrence of SCN- as environmental factor, its alimentary significance and its vitalizing effect (stimulation of nonspecific and specific warding off, stimulation of proliferation, protective effect at toxic loading) are of interest for the fundamental and applied research.

[Status of immunological reactivity in workers engaged in the production of thiourea, ammonium rhodanate and cobalt and manganese salts]. / Sostoianie immunnoi reaktivnosti rabochikh proizvodstva tiomocheviny, rodanistogo ammoniia, solei kobal'ta i magrantsa.

The proposed study of the immunologic reactivity state in chemical plant workers revealed humoral and cellular reactivity changes caused by chemical agents and the workers' immunologic background. It was established that cobalt and manganese salts caused initial immune response.

Allergic contact dermatitis to nasturtium.

In conclusion, oil of mustard, contained in many plants and recognized mainly as a skin irritant, is also capable of causing an allergic contact dermatitis. Nasturtium, which contains mustard oil, should be added to the list of plants capable of causing this dermatitis and must be suspected in any patient who handles plants and presents with hand dermatitis.

Hierarchy of cytotoxic T cell clones. II. Intraclonal triggering of lysis by hapten-specific CTL.

Cells from clones of anti-hapten murine cytotoxic T lymphocytes (CTL) can act as both target and effector cells, but will not lyse members of the same clone. The effect of haptenation on the cytolytic activity of anti-fluorescein (FL) and anti-trinitrophenol (TNP) CTL clones was examined. Treatment of anti-FL clones with fluorescein isothiocyanate or anti-TNP clones with trinitrobenzene sulphonic acid induces these clones to kill in an antigen-independent fashion. Targets killed by the haptenated CTL included syngeneic and allogeneic B lymphocyte blast cells, P815, YAC-1 and in one case human GM 4072 tumor cells. The importance of CD8 and T cell receptor (TCR) occupancy is demonstrated by the ability to block autotriggering by antibody directed against Ly 2 and the TCR. The results demonstrate that effects other than antigen recognition of the target play a role in the final outcome of effector-target cell interactions and provide a mechanism which could lead to autodestruction and immunosuppression particularly in some types of viral infection.

Physiology of murine B lymphocytes. I. Life-spans of anti-mu and haptenated Ficoll (thymus-independent antigen)-reactive B cells.

We have evaluated the life-span of B lymphocytes by measuring the functional reactivity of normal B cells upon transfer into xid mice, which do not respond to anti-mu, fluoresceinated-Ficoll (FL-Ficoll) and 2,4,6-trinitrophenyl aminoethylcarbamylmethyl Ficoll (TNP-Ficoll). After 4 days of transfer only 30-40% of anti-mu-reactive cells decayed leaving behind 60-70% of B cells which appeared to decay slowly. Even 10 days after transfer approximately 40% of anti-mu-reactive B cells can be recovered from the recipients. This result demonstrates the existence of heterogeneity in the life-spans of anti-mu-reactive B lymphocytes and that a major population (60-70%) of B cells persists beyond 4 days. Interestingly, the short-lived B cell subpopulation was not detected when the decay of the antigen-specific B cells was studied. Thus, TNP-Ficoll and FL-Ficoll-reactive B cells were found to be long lived and such B cells did not decline at all, even 5 months after transfer into xid mice.

An immunocytochemical method for the detection of fluorochrome-labelled DNA probes hybridized in situ with cellular RNA.

Various detection systems for in situ hybridization of nucleic acids are currently used. We report here an immunocytochemical detection system which is based on the detection of FITC-labelled DNA/mRNA hybrids and takes advantage of FITC molecules attached covalently to the DNA probes prior to hybridization. In situ hybridization on cytocentrifuge spots is followed by the application of an anti-fluorescein antibody thus permitting detection of mRNA/DNA-FITC hybrids. The anti-FITC antibody reaction is demonstrated by an indirect immunocytochemical peroxidase-staining method. The T lymphoblast cell line Jurkat and the cDNA for the TCR-beta chain were chosen to establish the technique.

Immunoadsorption of T cells onto glass beads using tetramolecular complexes of monoclonal antibodies.

Tetramolecular monoclonal antibody complexes were used to selectively cross-link a subset of human peripheral blood T cells (CD8 positive) to glass beads coated with hapten (fluorescein) modified bovine serum albumin. Tetramolecular antibody complexes were prepared with anti-CD8 (Leu 2a), anti-FITC mouse IgG1 monoclonal antibodies and monoclonal rat anti-mouse IgG1. Optimum conditions for depletion of CD8 positive cells from peripheral blood mononuclear cell suspensions were determined with 1 ml columns. 90-99% of the CD8 positive cells could be removed with 0-17% non-specific adsorption of CD8 negative cells. The weakest link in this system was the bond between hapten-modified albumin and the glass beads. These results indicate that tetramolecular antibody complexes are useful for the specific immunoadsorption of cells to a defined affinity matrix.

Blocking of acquisition and presentation of antigen by dendritic cells with cyclosporine. Studies with fluorescein isothiocyanate.

The effect of cyclosporine on the acquisition and presentation of antigen by dendritic cells (DC) was examined. Mice skin-painted with the contact sensitizer FITC received 100 mg/kg of CsA orally on the day before and the day of sensitization. This blocked the development of delayed hypersensitivity as measured by ear-swelling on challenge with antigen on day 6. The antigen-presenting DC in the draining lymph nodes 24 hr after skin painting were studied. The numbers of DC within the lymph node more than doubled after skin painting and this was not altered by the treatment with CsA. The DC from normal skin-painted animals showed a biphasic distribution of antigen, with more than half the cells acquiring high levels of antigen and the remainder having low amounts. In the animals treated with CsA most cells had low levels of antigen. The DC from untreated animals stimulated primary proliferative responses of syngeneic lymphocytes in vitro and initiated delayed hypersensitivity in recipient animals, but the DC from CsA-treated animals did not stimulate immune responses. Cyclosporine may, therefore, prevent acquisition and presentation of antigen by DC in addition to any direct effects on T and B cells.

The production and radioimmunoassay application of monoclonal antibodies to fluorescein isothiocyanate (FITC).

Monoclonal antibodies (MoAbs) were produced against the fluorescence marker fluorescein isothiocyanate (FITC). FITC was used as a hapten to label different proteins and the anti-FITC MoAbs were used to identify these labelled proteins in a solid-phase radioimmunoassay and in cellular radioimmuno-binding assays for the demonstration of antigens and antibodies.

Production and ELISA application of bispecific monoclonal antibodies against fluorescein isothiocyanate (FITC) and horseradish peroxidase (HRP).

Hybrid hybridomas producing bispecific monoclonal antibodies reacting with both horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC) were obtained by fusing two hybridoma lines and selecting the fused cells using a fluorescence activated cell sorter (FACS). FITC was used to label different monoclonal antibodies and the bispecific antibodies acted as a linking agent between FITC-labelled antibody and the marker enzyme HRP. This system was used in enzyme immunoassays for the detection of different antigens. The results suggest a wide application of bispecific anti-FITC/anti-HRP antibodies as a detection system in EIA.

A sensitive method of detecting proteins on dot and Western blots using a monoclonal antibody to FITC.

Monoclonal antibodies to FITC were produced and shown to be specific for the fluorochrome. Molecular weight marker proteins labelled with FITC could be detected after SDS-PAGE and transfer onto nitrocellulose using anti-FITC followed by an anti-mouse IgG-alkaline phosphatase conjugate. The molecular weight of an antigen common to Legionella pneumophila and recognised by a monoclonal antibody could be determined accurately on a Western blot when FITC labelled markers were used as internal standards. The FITC-anti-FITC system was shown to be extremely sensitive, detecting 23.7 amol of BSA-FITC conjugate (equivalent to 1.42 x 10(7) molecules of FITC) in a dot blot assay.

Regulation of B-cell expression as revealed in the congenic barrier: exerted by B cells, augmented by T cells, and directed toward memory as well as naive donor B lymphocytes.

Donor's memory cell expression is reduced in nonirradiated recipients as compared to irradiated ones. This "barrier" was studied in Igh-congenic BALB/c mice as transfer partners, making use of their allotypic markers, under two aspects: (I) Does the barrier apply to naive B-cells also, not only to memory cells? (II) Does a barrier exist in nude recipient mice also? (I) Naive donor B cells were subject to a barrier in nonirradiated recipients as well as memory cells. (II) Nude recipients showed a barrier toward naive donor cells like euthymic hosts, but toward memory cells this was less stringent. The allotypically marked donor B cells survived and were active in the nonirradiated host. In some situations they even dominated the host's response. The results favor a concept of the barrier as an "intrinsic" interlymphocytic control, exerted by B cells, augmented by T cells, best explained by Jerne's theory of idiotypic interactions.

Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. Functional and morphological studies.

We have examined the cells involved in the development of contact sensitivity to FITC in CBA mice. After skin painting with antigen, the number of dendritic cells (DC) in the draining lymph nodes increased by 30 min, was maximal at 48 h, and returned to normal by 6 d. Derivation of some DC from Langerhans' cells of the skin was indicated from the presence of Birbeck granules observed in some DC isolated 24 h after skin painting. The DC acquired FITC and by 8 h there were two populations, one highly fluorescent and the other less fluorescent. The highly fluorescent cells were present between 8 h and 3 d after sensitization, and during this period the DC were potent at initiating primary proliferative responses of normal syngeneic T lymphocytes in vitro. Between days 3 and 5 the numbers of lymphocytes in the draining lymph node increased. During this period purified T lymphocytes did not express detectable levels of antigen, but enriched B cell populations expressed antigen transiently on day 1, 2, or 3 after exposure to antigen. The results showed that, during a 3-d period after exposure to antigen, DC expressed antigen and stimulated T cell proliferation. We speculate that low amounts of FITC binding selectively to veiled cells or lymph node DC in the first hours after exposure to antigen are not immunogenic but that Langerhans' cells acquire high levels of antigen, enter the nodes, and initiate immune responses.

The effect of divalent and univalent binding on antibody titration curves in solid-phase ELISA.

This paper describes the influence of antigen coating concentration, epitope density per antigen molecule and anti-immunoglobulin reagents on antibody titration curves in solid-phase ELISA. Based on results obtained with fluorescein as the hapten and monoclonal anti-fluorescein antibody, which were confirmed in another antigen-antibody system, it is concluded that: (a) Antibody titration curves are independent of antigen-coating concentration in a limited range of concentrations only. (b) The complex between one antibody and two epitopes ('divalent binding') is more stable than the complex between one antibody and one epitope ('univalent binding). The ratio between divalent and univalent binding depends on the epitope density per antigen molecule and on the antigen-coating concentration. (c) The prozone phenomenon can be explained by an increased instability of plate bound antibodies due to a shift from divalent to univalent binding. (d) In solid-phase ELISA a correct evaluation of the antiserum specificity can be performed only if it is ascertained that all target antigens are coated under saturating conditions.