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.

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.

Antigen valence determines the binding of nominal antigen to cytolytic T cell clones.

We have shown that cytotoxic T cell clones specific for the nominal antigen FL will bind high molecular weight (600,000 to 2,000,000) polyacrylamide and Ficoll polymers conjugated with 200-600 FL groups per molecule. Low molecular weight polymers (40,000) with the same epitope density did not give stable binding. A high molecular weight polymer with a lower epitope density also failed to bind. Taken together, these results suggest that a substantial degree of multivalence is a necessary factor in the stable binding of nominal antigen to T cell clones.

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.

Induction of immunological tolerance requires that the B cells can respond to the polyclonal B-cell-activating properties of the thymus-independent antigens.

Mice were rendered specifically tolerant to the fluorescein isothiocyanatedextran (FITC) epitope by injection of FITC-dextran B512. Their spleen cells were removed at various times and cultivated in vitro with different polyclonal B-cell activators, such as lipopolysaccharide (LPS), purified protein derivative of tuberculin, and native dextran. LPS caused the appearance of high affinity anti-FITC plaque-forming cells to an equal extent with cells from untreated and tolerant animals, whereas native dextran failed to activate cells from tolerant mice, although it was a potent activator of normal cells. It was concluded that tolerance induction only affects those B cells that could respond to the polyclonal B-cell-activating properties of the tolerogen, but not other B cells having an identical set of Ig receptors directed against the tolerogen.

Idiotypic cross-reactivity of low-affinity anti-fluorescyl monoclonal antibodies.

Previous studies concerning structure-function relationships of anti-fluorescyl hybridoma proteins utilized primarily high-affinity proteins (Ka greater than 5.0 X 10(7) M-1) possessing distinct idiotypes. Low-affinity anti-fluorescyl monoclonal antibodies, predominantly IgGl or IgG2a possessing kappa light chains were analyzed. Two fusions produced 18 monoclonals, 13 binding fluorescein with a low affinity (less than or equal to 3.0 X 10(6) M-1) and five possessing high affinities (greater than or equal to 5.3 X 10(8) M-1). Solid-phase idiotype assays, utilizing rabbit anti-idiotype reagents against two low-affinity proteins (3-13 and 3-17), showed that all the low-affinity clones (except 2-9 and 2-21) were capable of inhibiting (40-100%) these two idiotype-anti-idiotype interactions while no high-affinity proteins inhibited them. The interactions with 3-13 and 3-17 were inhibited 100 and 88%, respectively, by free fluorescein. When these idiotype-anti-idiotype interactions were inhibited with increasing concns of heterologous hybridoma proteins, three clones inhibited both interactions as effectively as the homologous proteins at all concns tested and inhibition reached 100%. These three clones appeared to possess all the idiotopes that the anti-3-13 and anti-3-17 reagents detected on 3-13 and 3-17. Screening of eight high-affinity anti-fluorescyl proteins previously produced [Kranz and Voss, Molec. Immun. 18, 889-898 (1981a)] identified a single clone [20-4-4 (Ka = 5.0 X 10(7) M-1)] significantly inhibiting the 3-13 and 3-17 interactions (71.0 and 63.6%, respectively). In addition, recombination experiments utilizing H- and L-chains derived from three low-affinity and three high-affinity antibodies resulted in reformation of active sites in all six heterologous combinations when both chains were derived from low-affinity antibodies, and in only one of six combinations when both chains were derived from high-affinity molecules. Thus, the apparent lack of private idiotopes on clones 3-13 and 3-17 and the presence of these idiotopes (or cross-reactive ones) on 11 of 13 low-affinity antibodies and on one of 13 high-affinity antibodies may indicate that clones 3-13 and 3-17 are encoded by germline genes. The H- and L-chain recombination experiments indicated that the idiotype and affinity of parental molecules may be involved in H- and L-chain association.

Comparative properties of monoclonal antibodies comprising a high-affinity anti-fluorescyl idiotype family.

The T-dependent BALB/c murine immune response to fluorescein (F1) is characterized by structural heterogeneity at the protein level exemplified in part by a significantly wide range of affinities (Ka), and apparent lack of dominant idiotypes. In order to generate an idiotype family, xenogenic anti-idiotype (anti-ID) antibodies raised against anti-F1 monoclonal antibody (MCA) 4-4 (Ka = 1.7 X 10(10) M-1) were used in a solid-phase radioimmunoassay (SPRIA) to screen 68 anti-F1 hybridomas generated from multiple cell fusions for idiotypically related immunoglobulins. Four affinity-purified MCAs (designated 9-40, 10-25, 5-14 and 5-27) bearing 4-4 idiotypic determinants (ID 4-4) exhibited discrete isoelectric focusing spectrotypes (pI range = 6.8-7.7), significantly different fluorescence quenching values (38-95%) of bound ligand, binding affinities ranging from 3.3 X 10(7) to 5.3 X 10(8) M-1, similar active site inaccessibility to iodide, and closely related fine-specificity patterns for fluorescyl analogues. Idiotypic relatedness of each MCA to prototype 4-4 was quantitated by SPRIA, the results demonstrating that: each 125I-labeled MCA bound significantly to solid-phase anti-ID 4-4, and the concns of heterologous MCAs 9-40, 10-25 and 5-14 required for 50% inhibition of 125I-4-4/anti-ID 4-4 binding were comparable to homologous Ig protein. The finding that ID 4-4 bearing anti-F1 MCAs exhibit various binding properties and affinities is consistent with variable-region somatic diversification in anti-F1 affinity maturation.

Induction and suppression of contact sensitivity to fluorescein isothiocyanate (FITC).

Although both fluorescein isothiocyanate (FITC) and trinitrophenyl (TNP) covalently couple primarily to the epsilon-amino group of lysine residues of surface glycoprotein, FITC is structurally different from TNP. In this paper, we investigated the immune regulatory mechanisms in contact sensitivity (CS) to FITC by the administration of FITC and FITC-conjugated epidermal cells (FITC-EC) via various routes. Mice injected with FITC via i.p. or i.v. route did not induce CS but induced hyporesponsiveness to the following sensitization with FITC painting. Mice injected with FITC via s.c. route induced neither CS nor hyporesponsiveness to the following FITC painting. Administration of FITC via i.v. route was demonstrated to induce hapten-specific suppressor T cells. Inoculation of FITC-EC via s.c. or i.p. route induced CS, whereas injection of FITC-EC via i.v. route did not induce CS but induced hyporesponsiveness to the following FITC painting. The results are compared with the previous data obtained by trinitrobenzene sulfonate and trinitrophenyl-conjugated epidermal cells.

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.

Some methodologic aspects of the chromium chloride method for coupling antigen to erythrocytes.

The influence of various methodological variables on the CrCl3 technique for coupling antigen to the surface of erythrocytes has been investigated. Tests should be performed in protein- and phosphate-free medium. The CrCl3 stock solution should be stored at a pH of 5.0 and working dilultions prepared in acetate buffer (pH 5.5). The coupling procedure itself was performed as described by Goding (1976) with slight modifications. The relation between antigen and CrCl3 concentrations was found to be of crucial importance as excess of antigen inactivates CrCl3 whereas lack of antigen or excess of CrCl3, leads to spontaneous agglutination. A preliminary test based on determination of the minimum concentration of CrCl3 which produces spontaneous agglutination in the absence of antigen, and coupling if antigen is available, can be employed to predict the optimal concentration with sheep, human or chicken red blood cells. The pH-dependency of the coupling process is emphasized.

Cell sorting using a universally applicable affinity chromatography matrix: solid-phase anti-fluorescein isothiocyanate antibody.

Procedures are described for fractionating cells utilizing a universally applicable cellular affinity chromatography matrix. The affinity matrix consists of immunoabsorption purified goat anti-fluorescein isothiocyanate antibody coupled to large derivatized polyacrylamide beads. This matrix may, in principle, be used to isolate any cell subpopulation provided it has a fluorescein-labeled ligand on its surface. In this report the matrix was used to isolate viable purified fractions of mouse surface Ig-positive cells, Lyt1 cells, and mouse lymphocytes that bind the lectin soybean agglutinin. A preliminary experiment using the anti-FITC beads suggested that this technique can provide a fraction of cells enriched in antigen binding cells. Cell populations isolated by this technique retain their ability to respond to in vitro mitogen stimulation, as well as their ability to be maintained in cell culture following fractionation. Additional experiments using a column consisting of goat anti-rabbit Ig antibody coupled to the same support material are also reported.

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.

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.

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.

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.

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 donor cell type controls anti-hapten (fluorescein isothiocyanate) primary antibody response to hapten-modified syngeneic cells.

Hapten (fluorescein isothiocyanate, FITC)-sensitized syngeneic red blood cells (FITC-RBC) are exceptionally active for induction of anti-hapten primary antibody response, and FITC-modified syngeneic spleen cells depleted of RBC (FITC-SSC) are not immunogenic [4]. The present study has demonstrated that FITC-SSC injected simultaneously with FITC-RBC inhibit partially the anti-FITC response to the latter. Either the immunogenicity of FITC-RBC or the response-inhibiting activity of FITC-SSC was increased as the concentration of hapten-sensitizing cells was raised from 0.005 mg/ml to 2 mg/ml. The inhibition of anti-FITC response by FITC-SSC strictly required live donor cells, but was not dependent on T-cell activity of either the donor or recipient. Neither FITC-thymocytes nor the FITC-T-cell-rich fraction of SSC showed a definite activity for inhibition, whereas the FITC-B-cell-rich fraction of SSC acted very effectively. These results suggest that the primary anti-hapten antibody response to hapten-modified syngeneic cells is primarily controlled by antigen-bearing live donor cells of different cell types.

Ontogeny of the erythrocyte-dependent IgM antibody responses to cell membrane antigens.

Our previous experiments characterized the T-cell independent type 2 B-cell responses to cell membrane antigens that are controlled by two donor cell types with different antigen-presenting (AP) activities. We here report about the ontogeny of this novel type of responses with special reference to the mutual relation of the development among two AP activities and their acceptor functions. The responses of mice to H-2d antigens on allogeneic cells and hapten (fluorescein isothiocyanate) antigens on syngeneic cells were examined in parallel. The positive AP activity displayed by red blood cells (RBC) for induction of anti-hapten responses was fully developed in the fetus, although H-2d antigens on the RBC for induction of anti-H-2d responses was immature in mice under 7 days old. In contrast, the negative AP activity displayed by spleen cells (B cells) for inhibition of the RBC-dependent anti-hapten and anti-H-2d responses was first developed in mice about 3 weeks old. The B cell functions accepting the positive and negative AP activities were also matured by that time. The possible significance of these findings in the physiology and pathology of the unique responses was discussed.