Anti-human Lysozyme Research Articles

Immunohistochemical Detection of Macrophages in the Short-finned Pilot Whale ( Globicephala macrorhynchus) and Risso's Dolphin ( Grampus griseus)

Macrophages play a central role in the immune system, but few markers are available for their detection in cetaceans. The purpose of the present study, therefore, was to examine the cross-reactivity for two cetacean species (short-finned pilot whale and Risso's dolphin) of four anti-human antibodies (SRA-E5, AM-3K, EBM11 and anti-human lysozyme). The distribution of SRA-E5- and AM-3K-positive cells was similar, both antibodies labelling (1) many resident macrophages in the spleen, lymph nodes, liver, lung, kidney, intestine and dermis, and (2) exudate macrophages in the hepatic interlobular septa. Anti-human lysozyme antibody also labelled both resident and exudate macrophages. However, double immunohistochemistry showed that the majority of AM-3K-positive cells in the spleen and liver were also labelled by SRA-E5; on the other hand, anti-human lysozyme-positive cells did not always correspond with AM-3K-positive cells. Cetacean tissues contained no EBM11-positive cells. The study demonstrated the potential values of SRA-E5, AM-3K and anti-human lysozyme antibody for cetacean macrophage studies.

Development of a microparticle-enhanced nephelometric immunoassay for quantitation of human lysozyme in pleural effusion and plasma.

A microparticle-enhanced nephelometric immunoassay, based on polystyrene beads coated with antihuman lysozyme antibody, has been developed for lysozyme quantification in sera and pleural effusions. The standard curve extends from 0.58 mg/l to 18.75 mg/l and no antigen effect was observed. The results showed a good serial precision. The intra-assay precision (n = 20) expressed as CV was between 2.2 and 4.2 in three different concentrations. The inter-assay precision, with different calibration curves (n = 12) was between 6.4 and 7.1. The analytical assay showed a sufficient linearity (r > 0.999). There were no interferences either with haemoglobin (up to 4 g/l), lipids (up to 0.5%, expressed as 1% Lipofundina content), or bilirubin (up to 5 mg/dl). The analytical sensitivity was lower than 0.6 mg/l. The correlation with a Micrococcus lysodeikticus turbidimetric assay showed a correlation coefficient of 0.915. We have studied 92 patients with pleural effusion. In each case, pleural fluid adenosine deaminase activity and pleural fluid to plasma lysozyme ratio were determined. The lysozyme ratio showed similar clinical sensitivity and specificity as to adenosine deaminase.

Synthesis of type II cell lamellar body lysozyme-15 kD protein (lbl-15) by perfused rat lung.

Isolated alveolar type II pneumocytes of the rat have been shown to secrete a 14 to 15 kD protein that has some sequence homology and immunoreactivity with lysozyme. Using immunochemical analyses of rat lung subcellular fractions and 35S metabolic labeling of isolated perfused lung preparations, we studied the subcellular distribution and synthetic pathway for this protein. SDS-PAGE and Western blotting of lamellar bodies (LB) using a polyclonal anti-human lysozyme (anti-HLZ) demonstrated a single band at 15 kD that was significantly enriched over rat lung homogenates, isolated lysosomes, and type II cell lysates. This 15 kD protein isolated from LB by immunoprecipitation with anti-HLZ also demonstrated functional lysozyme activity and was termed lamellar body lysozyme (lbl-15). Analysis of LB and surfactant (SF) isolated from 12 separate perfused lung preparations labeled for 6 h with 35S-labeled amino acids demonstrated that lbl-15 represented a significant portion of the radiolabeled LB proteins (5.9% of total LB radioactivity). Lamellar bodies and an extracellular fraction (surfactant) obtained from rat lungs perfused with 35S-methionine-cysteine for varying times both showed time-dependent appearance of lbl-15. At all time points, the specific activity of lbl-15 was greater in LB than in SF. The kinetics for appearance of lbl-15 in LB and SF was similar to that for surfactant protein A. These results indicate that in the rat lung, type II cells synthesize a 15 kD protein (lbl-15) that is secreted into the alveolar space via an organellar pathway involving LB.(ABSTRACT TRUNCATED AT 250 WORDS)

High level expression of the synthetic human lysozyme gene in Aspergillus oryzae.

Aspergillus oryzae was transformed with a synthetic gene consisting of a chicken lysozyme signal sequence and a mature human lysozyme (HLY) sequence. The transformants secreted active HLY (about 1.2 mg/l) when the HLY gene was expressed under the control of the Taka-amylase A gene (amyB) promoter. Western blot analysis suggested that the secreted protein was immunoreactive with anti-human lysozyme antibody and the signal peptide was correctly cleavaged off in the A. oryzae transformants. The transcriptional level of the HLY gene was investigated by Northern blot analysis using a probe that was equivalently specific to both the HLY gene and the amyB gene. The HLY gene was expressed at a higher level compared with the amyB gene because of its multi-copy integration. The efficient transcription of the HLY gene suggested that A. oryzae is a promising host for production of heterologous proteins from higher eukaryotes.

Bactericidal activity of C9-deficient human serum.

Escherichia coli B/SM, strain 1-1, was killed dose dependently by human hereditary C9-deficient serum (C9DHS), which was shown to contain no C9 Ag by an ELISA method. On the other hand, human hereditary C7-deficient serum did not kill the bacteria under similar conditions. The bactericidal activity of C9DHS was inhibited by rabbit anti-C5 antibody but not by murine anti-C9 mAb. The anti-C9 antibody decreased the bactericidal activity of normal human serum (NHS) to the level of that with C9DHS. Sheep anti-human lysozyme antibody did not affect the bactericidal activity of C9DHS or NHS even when added at more than twice the concentration required to block the serum lysozyme activity on Micrococcus luteus. After treatment with C9DHS and washing, surviving Escherichia coli were killed by C9, but not by lysozyme, transferrin, or both. Other strains of E. coli (K12 W3110, C600, and NIHJ) and Salmonella typhimurium (strain NCTC 74), all maintained in the laboratory, were also killed by C9DHS. However, pathogenic strains recently isolated from patients with traveler's diarrhea and some strains of S. typhimurium were resistant to both C9DHS and NHS, at least at the serum concentration tested. A concentration of 0.1 M Tris did not increase the susceptibility of serum-resistant strains of bacteria to C9DHS, but made one strain of S. typhimurium tested susceptible to NHS, but not to C9DHS. These results clearly showed that C9DHS kills bacteria that are sensitive to NHS through activation of C up to the step of C8 in the same way that C9-deficient C serum lyzed sensitized erythrocytes.

De Novo Synthesis of Lysozyme by Human Epidermal Cells

The presence of lysozyme in human epidermis was determined in extracts from the surface of human skin and in sonicates of human epidermal cell preparations with the use of a bacteriolytic assay employing Micrococcus lysodeikticus cell walls as substrate. Preincubation of the extracts with the Fab portion of the IgG fraction of an antiserum to human lysozyme abolished the lytic activity of the extracts showing the specificity of the assay. De novo synthesis of lysozyme by human epidermal cells was demonstrated by radiolabeling studies. Human epidermal cells cultured in serum-free medium and pulsed with [3H]leucine for 24 h were sonicated and fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A major band at Mr 14,500 exhibited both immunoreactivity with a rabbit antihuman lysozyme antibody and radioactivity. Gel filtration of the cell sonicate revealed bacteriolytic activity in the fractions containing radioactive and immunoreactive proteins. These findings suggest that lysozyme is newly synthesized by human epidermal cells.

Killing of an encapsulated strain of Escherichia coli by human serum.

Changes in cell viability and in factors affecting metabolic integrity were examined after exposure of Escherichia coli LP1092 to human serum. Antibody-dependent classical pathway activity accounted for the rapid killing of strain LP1092 by complement. Removal of serum lysozyme by bentonite absorption or by neutralization with anti-human lysozyme immunoglobulin G resulted in a reduction in the rate of killing; optimal activity could be restored by the addition of physiological amounts of egg-white lysozyme. The pattern of 86Rb+ and alkaline phosphatase release obtained after serum treatment did not support the view that complement simultaneously disrupts cytoplasmic and outer membrane integrity. Macromolecular synthesis was affected late in the reaction sequence; complete inhibition of precursor incorporation into RNA, DNA, and protein occurred only after almost total loss of bacterial colony-forming ability. Addition of chloramphenicol, an inhibitor of protein synthesis, to the bactericidal system resulted in a marked reduction in the rate of serum killing. Killing was completely inhibited by an inhibitor (KCN) and an uncoupler (2,4-dinitrophenol) of oxidative phosphorylation. Exposure of LP1092 cells to serum was followed by a rapid and large increase in intracellular ATP levels; ATP synthesis did not occur when bacteria were exposed to dialyzed serum, which killed LP1092 cells at a much reduced rate. Addition of glucose or serum ultrafiltrate to dialyzed serum restored optimal bactericidal activity. We suggest that optimal killing of gram-negative bacteria is an energy-dependent process requiring an input of bacterially generated ATP.

A predominant idiotype on anti-hen egg white lysozyme antibodies from diverse mouses strains.

A predominant idiotype was detected on almost all antibody molecules directed against the multideterminant antigen, hen egg white lysozyme (HEL). This idiotypic set was defined by rabbit and quinea pig antiidiotypic antisera raised against a rare cross-reactive antibody population isolated and from a heterogeneous BALB/c anti-human lysozyme (HUL) humoral response. This isiotype was fond on the majority of all HEL-reactive antibody from all strain tested; its presence was independent of Ig-1 allotype of H-2 haplotype. The idiotype was also present on the majority of anti-HEL antibody from nonresponder mice immunized with keyhole limpet hemocyanin coupled to HEL. The idiotype was not found on normal mouse immunoglobulin or on antibodies specific to two other proteins, HUL and ribonuclease, which are similar to HEl in size and charge. Individual idiotype-positive anti-HEL antibody populations exhibited specificity differences when analyzed for reactivity toward other avian lysozymes, suggesting that distinct clonal products shared the idiotype specificities. Fractionation of heterogeneous anti-HEL antibodies by preparative isoelectric focusing yielded distinct antibody fractions that were demonstrated to share the relevant idiotype determinants. The data are discussed in terms of the HEL peptide specificites of idiotype-positive and -negative antibodies.

Sequential metabolic expressions of the lethal process in human serum-treated Escherichia coli: role of lysozyme.

Several metabolic parameters indicative of Escherichia coli function and integrity were kinetically examined in response to treatment with normal human serum in the presence and absence of functional human lysozyme. Specific inhibition of this enzyme in bacteriolytic and bactericidal reactions was accomplished by using purified rabbit anti-human lysozyme immunoglobulin G. Initiation of the complement-mediated alterations of cytoplasmic membrane integrity, as judged by the leakage of 86Rb from prelabeled cells or the hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by a cryptic strain, was found to be independent of lysozyme action. Furthermore, inhibition of macromolecular synthesis by E. coli in response to serum treatment occurred at the same time regardless of the functional state of lysozyme. Although the rate and extent of bacteriolysis were reduced in the absence of lysozyme, the bactericidal kinetics was unaffected. These results demonstrate that the lethal events associated with the action of antibody and complement on gram-negative bacteria are independent of lysozyme, suggesting an accessory role for this enzyme in immune reactions. A possible temporal sequence of complement-induced effects occurring at the cell surface is presented.

Modulation of neutrophil function by lysozyme. Potential negative feedback system of inflammation.

Host responses to infectious organisms should be modulated so that tissue-damaging products of inflammatory cells do not produce excessive destruction of normal tissue. Lysozyme, which is continuously secreted by monocytes, which, in turn, migrate relatively late to inflammatory areas, was found to significantly dampen several responses of neutrophils to inflammatory stimulants. Thus, human lysozyme obtained and purified from the urine of patients with monocytic leukemia (but not its structurally similar and comparably cationic analogue, eggwhite lysozyme) depresses chemotaxis of normal neutrophils to activated complement, bacterial supernate, and N-formylmethionyl-phenylalanine. In addition, human (but not eggwhite) lysozyme depresses oxidative metabolism (hexose monophosphate shunt activity) and superoxide generation of neutrophils. The specificity of the suppressive effects was indicated by inhibition studies with rabbit antihuman lysozyme antibody, and with the trisaccharide of N-acetylglucosamine, a specific inhibitor of lysozyme. The results suggest that lysozyme, a product of inflammatory cells themselves, may function in a negative feedback system to modulate the inflammatory response.

Further studies of an anomalous cross-reaction involving worm and vertebrate lysozymes

Lysozyme was purified from the annelid worm Nephthys hombergii and re-examined for immunological cross-reactivity with antisera to human and chicken lysozymes. It was determined that the previously reported reaction between worm lysozyme and anti-human lysozyme and also the presently observed reaction between worm lysozyme and anti-chicken lysozyme were due to the presence of small amounts of vertebrate-like c-type lysozymes in the worm enzyme preparations. The cross-reactions were not attributable to the major lysozyme component in the worm preparations.

Immunologic detection of intracellular and cell-surface lysozyme with human and experimental leukemic leukocytes

Intracellular lysozyme (muramidase) was detected by both indirect and direct immunofluorescent microscopy in human and rat leukocytes by labeling methanol-fixed cells with the globulin fraction of immune rabbit serum prepared against the homologous lysozyme. Normal human polymorphonuclear leukocytes (PMNLs), monocytes, and cells from patients with chronic myelogenous leukemia, acute myelogenous leukemia, and acute monomyelogenous leukemia were lysozyme positive, while lymphocytes, basophils, eosinophils, and cells from patients with acute lymphatic leukemia were negative. Similarly, normal rat PMNLs, monocytes, and cells from three transplantable rat myelogenous leukemias were positive in the rat lysozyme assay, while normal rat lymphocytes and spontaneous acute mononuclear cell leukemias of two inbred strains were negative. By labeling viable cells with the purified F(ab 1) 2 fragment of anti-human lysozyme immunoglobulin, lysozyme could be detected on the surface of cells and surface-lysozyme-positive cells could be separated from other human bone marrow leukocytes using a fluorescence activated cell sorter. Lysozyme appears to be a potentially valuable cell-surface marker for identification and separation of myeloid, monocytic, and histiocytic cells.

Ultrastructural immunocytochemical localisation of lysozyme in human bronchial glands.

An immunocytochemical unlabelled antibody method using rabbit antihuman lysozyme, antirabbit immunoglobulin, and soluble rabbit antihorseradish peroxidase/horseradish peroxidase complexes was used to study the fine structural distribution of lysozyme in human bronchial glands. None was identified in mucous cells but there was heavy staining of the serous cell granules. The serous cell granules were not stained uniformly, suggesting the presence of other secretory products but lysozyme secretion appears to be a major function of these cells. The pathological implications of this are discussed.

Cross-reactivity of some rabbit anti-human lysozyme sera with gallinaceous lysozymes

Sera from several rabbits immunized with human lysozyme were found to agglutinate hen egg-white lysozyme-coated sheep red blood cells. When assayed for direct binding with 125I-labeled hen egg-white lysozyme, these sera were found to give approximately 5–10% of the binding found with 125I-labeled human lysozyme. This binding was inhibitable by unlabelled human lysozyme. Binding similar to that of hen egg-white lysozyme was found with Japanese quail lysozyme. However, 125I-labeled turkey egg-white lysozyme was found to be bound to a greater extent. An explanation offered for the greater cross-reactivity of turkey lysozyme with anti-human lysozyme, as compared to that found with hen or Japanese quail lysozyme, invokes the presence of Leu instead of His at position 15 in turkey and human lysozymes.

Structural basis for immune recognition of lysozymes-IV. Immunologically active peptide obtained by the action of cyanogen bromide on human lysozyme

The two methionyl peptide bonds of lysozyme of human origin were cleaved by cyanogen bromide in 70 per cent formic acid. Cleavage was shown by the absence of methionine and the appearance of homoserine and homoserine lactone on amino acid analysis, detection of two new amino groups, and separation of the expected peptides upon Sephadex gel filtration. The products were shown to consist of a free single polypeptide chain (residues 18–29) and a remaining molecule with two polypeptide chains (residues 1–17 and 30–129) held together by 4 disulfide bridges. The enzymatic activity of the cyanogen bromide treated lysozyme or of its products was destroyed. Immunological studies revealed that both CNBr products can react with antibodies prepared against native lysozyme. This was demonstrated by their ability to; (a) inhibit the inactivation of human lysozyme-T 4 bacteriophage conjugate; (b) to bind specifically to antibody-Sepharose conjugate and (c) to cause inhibition of the binding of radioactively labelled human lysozyme by anti-human lysozyme serum.