Separation of phagocytic leukocytes from the peripheral blood of chickens

Phagocytic leukocytes were separated from the peripheral blood of chickens using a one-step Percoll density gradient technique. Heterophils recovered from two fractions of the gradient were of 96.9 to 99.8% purity and were fully viable and functional, as demonstrated by their capacity to phagocytose latex beads and staphylococci. Adherent mononuclear cells were cultured from specific gradient fractions and shown to phagocytose staphylococci.     

Changes in DNA and surface properties of peripheral blood leukocytes in monkeys after whole-body γ-irradiation

Central Roentgeno-Radiologic Research Institute, Ministry of Health of the USSR, Leningrad. Institute of Biophysics, Ministry of Health of the USSR, Moscow. Research Institute of Experimental Pathology and Therapy, Academy of Medical Sciences of the USSR, Sukhumi. (Presented by Academician of the Academy of Medical Sciences of the USSR V. A. Lapin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 8, pp. 158–159, August, 1989.     

Ultrastructural localization of peroxidatic catalase in human peripheral blood leukocytes.

Localization of peroxidatic catalase in human peripheral blood leukocytes was accomplished by the assessment of alkaline diaminobenzidine reaction in the cytoplasmic granules of normal and acatalasemic leukocytes. A modified cytochemical procedure of Novikoff and Goldfischer (Novikoff AB, Goldfischer S: J Histochem Cytochem 17:675, 1969) and of Fahimi (Fahimi HD:J Cell Biol 43:275, 1969) was employed to improve the specificity of alkaline diaminobenzidine test for catalase. Diaminobenzidine-positive reaction for peroxidative catalase was observed in large and medium-sized granules in the cytoplasm of normal neutrophils, but a striking and notable absence of this reaction was observed in acatalasemic neutrophils. The test for myeloperoxidase, with the diaminobenzide reaction performed at neutrality, disclosed positively stained granules in both normal and acatalasemic neutrophils. Similarities in size and configuration of the positively stained granules for these enzymes suggest that catalase is sequestered in organelles which may be primary or azurophilic granules. Myeloperoxidase has been shown to be localized in the primary granules by others. It is possible that catalase and myeloperoxidase may be sequestered together or separately in these granules, but the present data do not permit us to draw this distinction. The ultrastructural localization of peroxidatic catalase and myeloperoxidase has been attempted in eosinophils, lymphocytes, and platelets, and the observations are compared with those of neutrophilic granules. The localization of peroxidatic catalase in monocytes could not be assessed satisfactorily because of the difficulties encountered in proper sampling of these cells.     

Neutral endopeptidase activity in human peripheral blood leukocytes

Neutral endopeptidase (NEP; EC 3.4.24.11) efficiently hydrolyses many neuropeptides. To determine the distribution of NEP, a possible regulatory enzyme for the neuropeptide-induced leukocyte activation, among human leukocytes, we investigated the enzymatic activity of NEP in each cell type of human peripheral blood leukocytes. The activity of NEP assessed by an NEP inhibitor phosphoramidon-sensitive Met5-enkephalin degrading activity was present in neutrophils (59.0 +/- 9.1 pmol/min 10(6) cells); however, the NEP activity was virtually absent in mononuclear cells, eosinophils and basophils. Common acute lymphoblastic leukemia antigen (CALLA) detected immunocytochemically with three anti-CALLA antibodies, whose amino acid sequence has been shown to be identical with that of NEP, was also found only in neutrophils, but not in other blood leukocytes. It is suggested that NEP might regulate the neuropeptide-induced activation of human neutrophils.     

Cultivation of human peripheral blood leukocytes in agar gel

The use of a modified method of cloning hematopoietic cells in semisolid nutrient media showed that among human peripheral blood leukocytes there are committed precursor cells of the granulocytic series (CFU_c). In healthy adults and children (aged from 4 days to 10 years) the number of these precursors is 0.05–6.38 and 0.2–2.9/10⁵ nucleated cells respectively. The number of CFU_c in the blood of patients with infectious mononucleosis is within normal limits (0.5–14/10⁵ nucleated cells). In acute leukemia very low colony-forming activity of the nucleated blood cells is found (0–0.3/10⁵ cells).Laboratory of Culture and Transplantation of Bone Marrow, Central Institute of Hematology and Blood Transfusion. Laboratory of Pediatric Hematology, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences, of the USSR N. A. Fedorov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 5, pp. 635–636, May, 1976.     

Cytotoxicity of human peripheral blood and colostral leukocytes against Shigella species.

We examined the ability of human peripheral blood leukocytes to kill strains of Shigella sonnei and Shigella flexneri by using a modified bactericidal assay. Antibody-dependent cellular cytotoxicity (ADCC) was demonstrated in the presence of specific rabbit immune serum directed against S. sonnei. With peripheral blood leukocytes from adults, ADCC was found only in the mononuclear cell and purified lymphocyte populations. Monocyte-macrophages and polymorphonuclear leukocytes were unable to demonstrate ADCC. Lymphocyte ADCC, which was not affected by the addition of phenylbutazone (an inhibitor of phagocytosis), was mediated by a non-T, Fc receptor-positive, HNK-1- cell. ADCC (using antiserum directed against virulent S. sonnei) was demonstrated against virulent S. sonnei but not against virulent S. sonnei or virulent S. flexneri. In contrast to leukocytes from adults, both mononuclear and polymorphonuclear cells from neonatal cord blood and from a patient with chronic granulomatous disease mediated anti-Shigella ADCC. Breast milk leukocytes (BMLs) collected 1 to 3 days postpartum were used as effector cells against virulent S. sonnei. The entire BML population, BMLs which did not adhere to plastic and BMLs which passed through nylon wool columns mediated both natural killer cytotoxicity and ADCC. In paired experiments, natural killer cytotoxicity and ADCC were significantly lower (30 to 45% inhibition) but not ablated, when phenylbutazone was added to BMLs and nylon wool-purified BMLs (P less than 0.05). These experiments suggest that colostral leukocytes mediated both extracellular and intracellular bacteriolysis in the presence and absence of specific antiserum. These mechanisms may be active in vivo in protection against shigellosis.     

Cathepsin D activity in human peripheral blood mononuclear leukocytes

To investigate the cellular origins of cathepsin D (CD) in inflammatory lesions, the CD content of lymphocyte subsets, monocytes, and macrophages were compared. Human monocytes, B lymphocytes, CD4+ T lymphocytes, and CDS + T lymphocytes were separated from peripheral blood of normal donors. CD content was 0.13±01g equivalents of CD per million cells and significant differences between different cell types were not found. To determine the CD content of macrophages, differentiation of peripheral blood monocytes was induced by either in vitro culture or treatment with 4-phorbol-12-myristate-13-acetate (PMA). Macrophages induced by five-day culture contained four times more CD than unstimulated monocytes, and macrophages induced by 18-h treatment with 20 mg/ml 4-PMA contained nine times more CD than monocytes treated with 4-PMA, an inactive stereoisomer of 4-PMA. These results suggest that macrophages are one of the enriched sources of CD in inflammatory lesions.     

Direct demonstration of engraftment of human peripheral blood leukocytes in SCID mice.

One of the major problems using man-mouse chimeras is still the difficulty to demonstrate unequivocally engraftment of human cells in murine tissues. Using supravital labelling of human leukocytes with the Hoechst dye H33342, it was possible to demonstrate directly their engraftment and to assess their distribution in the tissues of the severe combined immunodeficiency (SCID) mice. The human cells can be traced for a period of 4-5 weeks. In contrast to earlier reports, combined marker and labelled-cell studies suggest that T-cell surface marker CD3 with reported specificity for human lymphocytes are indeed found, also in man-mouse chimeras, only on human cells. The ratio of B and T cells of human origin changes significantly after transfer into SCID mice and differs among various SCID tissues. The simple staining procedure using the supravital nuclear dye H33342 opens new possibilities for the study of cellular interactions and host responses of the human immunoreactive cells in an increasingly well-characterized animal model.     

Fungicidal activity of human peripheral blood leukocytes against Paracoccidioides brasiliensis yeast cells.

Although epidemiological findings suggest that normal humans are resistant to Paracoccidioides brasiliensis infection, the host defense mechanisms against this fungus have not been fully understood. Here we examined human leukocytes for antifungal activity against yeast cells of this fungus, using an improved mycological culture medium with high plating efficiency for the yeast cell. In an attempt to minimize the impairment of leukocyte activities during the isolation process, leukocytes removed by centrifugation from a buffy coat of peripheral blood were used in the antifungal assay without further fractionation. The leukocytes thus prepared effectively killed P. brasiliensis yeast cells within the first 4 h of co-culture. Adding interferon-gamma (37 ng/ml), granulocyte-macrophage colony-stimulating factor (5 ng/ml), interleukin (IL)-1beta (12 ng/ml), or IL-4 (12 ng/ml) to the assay system enhanced the leukocyte antifungal (growth inhibitory) activity by 48 h. By contrast, addition of IL-8 (50 ng/ml) impaired the leukocyte activity. Tumor-necrosis factor-alpha (50 ng/ml) or IL-10 (25 ng/ml) had no effect in this respect. Dexamethasone (1 micromol/l) reduced the antifungal activity of leukocytes. This is the first demonstration that human leukocytes are able to effectively kill yeast cells of P. brasiliensis.     

Group A streptococcal peptidoglycan-polysaccharide inhibits phagocytic activity of human polymorphonuclear leukocytes.

Injection of sterile aqueous preparations of the peptidoglycan-polysaccharide of group A streptococci (PG-APS) produces chronic inflammation in several animal models. Chronic bacterial infection may be involved in some aspects of the pathogenesis of inflammation associated with the accumulation of PG-APS. Accordingly, the effect of PG-APS on human neutrophil (polymorphonuclear leukocyte [PMN]) bactericidal activity was studied with the supposition that this interaction may contribute to the inflammation observed. Concentrations of PG-APS greater than 10 micrograms/ml inhibited the ability of PMNs to kill Staphylococcus aureus. This inhibition was not due to a cytotoxic effect of PG-APS on PMNs, nor did PG-APS inhibit PMN metabolism required for the formation of microbicidal oxygen reduction products. PG-APS concentrations of 10 micrograms/ml or greater in the presence of 10% normal serum inhibited the attachment of bacteria to PMNs by 49% as compared with control cell populations. The concentrations of PG-APS required to inhibit uptake of Staphylococcus aureus were identical to those required for inhibition of PMN bactericidal activity. This inhibition did not occur in the presence of serum-free medium or medium with sera that had been heated to inactivate complement. These results show that PG-APS interacts with serum to inhibit PMN-mediated killing of S. aureus, most probably by interfering with bacterial uptake.     

Entry of adult T-cell leukemia-associated virus into human peripheral blood leukocytes

Summary Within 10 minutes after tritium-labeled adult T-cell leukemia-associated virus (ATLV) inoculation, silver grains were found over human lymphocytes. At the time of entry of ATLV, the viral envelope was observed to fuse with the cell membrane.With 3 Figures     

Cell suppression in PPD-induced blast specific response of human peripheral blood lymphocytes.

Secific stimulation of T-cells by PPD was inhibited by their autologous B cells. This inhibition was obtained with B cells separated either by depletion of E-RFC or by elution, with human IgG of lymphocytes bound to Sephadex beads coated with rabbit antibodies anti-human Fab fragments. The suppression was proportional to the number of B cells added to 10(6) T cells incubated with PPD and as previously reported was more marked in the case of B or T cells from BCG-vaccinated subjects with negative skin tests. The suppressive phenomenon required viable B cells and was inhibited by cycloheximide but was not altered by pretreatment of suppressor cells with actinomycin D or colchicine. It seems that B-suppressor cells interfere with recognition of PPD by T cells rather than with the proliferative phase of the specific blast response. Using various surface markers (i.e. Ig, C3 and Fc receptors) it was shown that the suppressor cells represent a subset of Ig-bearing B cells which do not carry Fc receptors.     

Characterization of interferons induced by bacteria and interferon-producing leukocytes in human peripheral blood.

Indirect evidence suggests that immunoglobulin A1 (IgA1) proteases may be factors in the pathogenesis of certain infectious diseases, including meningitis, gonorrhoea, and destructive periodontitis. Bacterial IgA1 proteases are therefore potential candidates as vaccines. In this study, IgA1 proteases from 166 clinical isolates and reference strains of Haemophilus influenzae and Haemophilus aegyptius were compared with regard to specific activity and pattern of enzyme inhibition by antisera raised against IgA1 protease from nine selected strains of H. influenzae. A total of 93% of H. influenzae strains and all H. aegyptius strains had detectable IgA1 protease activity. The majority of strains cleaved a prolyl-seryl or a prolyl-threonyl peptide bond in the alpha 1 hinge region, whereas occasional H. influenzae strains possessed two separate IgA1 proteases with these two specific activities. Of the 155 IgA1 protease-producing strains, all except 12 could be assigned to one of 14 IgA1 protease "inhibition types," each defined by a characteristic pattern of inhibition by the nine antisera. There was no correlation between IgA1 protease type and biotype of the strains. However, among 92 encapsulated H. influenzae strains, a close correlation between capsular serotype and IgA1 protease type was observed. With the exception of serotype f, strains of all capsular serotypes produced an exclusive antigenic type of IgA1 protease. All 38 strains of serotype b produced IgA1 protease of inhibition type 1, which was never demonstrated in non-encapsulated H. influenzae strains. These results facilitate the detection of an antibody response against specific IgA1 proteases and are of practical value for a possible future vaccine against H. influenzae serotype b infections.     

IgG4 and release of histamine from human peripheral blood leukocytes

Allergen-specific IgG4 was found in the sera from many normal, nonallergic individuals. Basophil leukocytes from donors with IgG4 antibodies to an allergen, but without IgE antibodies to the same allergen, did not release histamine when challenged with this allergen. In some cases, anti-IgG4 antiserum induced a release of histamine from the leukocytes. However, these cells also release histamine after incubation with normal rabbit serum or normal sheep serum. It is concluded that the IgG4-RAST cannot be used for the detection of IgG short-term sensitizing antibodies.     

Enhanced phagocytic activity of blood leukocytes in athymic nude mice

After 30-min incubation, blood leukocytes of adult athymic BALB/c nude mice (nu/nu) have a distinctly higher methacrylate or yeast particle uptake than leukocytes of euthymic nu/+ or +/+ mice. This permanent enhancement is not due to humoral factors, since the percentage of phagocytosing nu/nu leukocytes increases further in nu/+ littermate's plasma. Also, chronic infection or intraperitoneal immunization causes an additional transitory increase of the percentage of phagocytosing leukocytes and numbers of particles ingested; the phagocytic performance of leukocytes of euthymic mice is raised under these conditions by a greater factor. In fetuses enhanced phagocytosis of nu/nu mice is found only in monocytes. The bulk of ingestion of methacrylate particles is mediated by Fc receptors and significantly more receptors for IgG2B, IgG1, and IgG2A are demonstrable on nu/nu neutrophils; ingestion of particles via these receptors is again higher in nu/nu neutrophils, whereas nu/nu monocytes display a higher uptake via Fc(IgG1) receptors.     

Histamine release from human peripheral blood leukocytes analyzed by histamine radioimmunoassay

Histamine release from human peripheral blood leukocytes, afterin vitro challenge with allergen extracts, is usually measured by fluorometry. In the present study we compared the results of the automated fluorometric histamine assay (Siraganian) with those of the Immunotech histamine radioimmunoassay. Histamine release dose — response curves obtained after histamine measurement by both methods were superimposable.     

Indirect blastogenesis of peripheral blood leukocytes in experimental gingivitis.

The blastogenic response of peripheral blood leukocytes to lipopolysaccharide (LPS) was followed over a short course of experimental gingivitis, developed in human volunteers who strictly avoided oral hygeine procedures for periods up to 9 days. Eleven young males initially received thorough dental prophylaxes and supervised oral hygeine until they acquired optimal gingival health. At this point, leukocytes (5 X 10(5)) incubated with 1.5 to 25 mug of LPS in serum-free media showed no response as measured by tritiated thymidine uptake. Coincubation of cells with LPS and phytohemagglutinin (PHA), however, caused synergistic enhancement of blastogenesis in every LPS-PHA dose combination tried. With progressive accumulation of dental plaque and the concomitant development of gingival inflammation, this synergistic response was lost and replaced, proportionately, by a direct response to LPS. The leukocyte response to PHA was marginally enhanced with gingivitis.