Monoclonal antibodies to human polymorphonuclear leucocyte granule antigens (original) (raw)
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Infection and Immunity, 1988
Uropathogenic strains of Escherichia coli bearing mannose-sensitive (type 1) fimbriae promote a unique pattern of degranulation from human polymorphonuclear leukocytes (PMN). Significant quantities of the primary (1 degree) and tertiary (3 degree) granule markers, neutral protease-myeloperoxidase and N-acetyl-beta-D-glucosaminidase, respectively, were released by PMN in a dose- and time-dependent manner when stimulated by these defined bacterial strains. Organisms bearing mannose-resistant (P) fimbriae promoted release of only the secondary (2 degree) granule marker, vitamin B12-binding protein. When this pattern of degranulation was compared to that produced by PMN in response to a variety of soluble and particulate stimuli, only the calcium ionophore A23187 similarly triggered 1 degree and 3 degree granule marker release. All the other stimuli tested--zymosan, serum-treated and unopsonized; n-formylmethionyl-leucyl-phenylalanine; and phorbol myristate acetate--promoted release of ...
Granule release by polymorphonuclear leukocytes treated with the lonophore A23187
The Anatomical Record, 1977
Polymorphonuclear leukocytes (PMN's) incubated three to eight minutes a t 37OC in medium containing 1 x M of the ionophore antibiotic A23187 released their cytoplasmic granules into the extracellular medium. Transmission electron microscopy of treated cells showed microfilament bundles extending between adjacent granules within the cytoplasm and between granules and the plasma membrane. Tiny dense projections (beads) 8-12 nm in diameter were observed along segments of the cytoplasmic surface of the plasma membrane with a periodicity of 20-30 nm. These beads were observed on the plasma membrane only in the vicinity of intra-or extracytoplasmic granules. The structural relationships of the beads with the plasma membrane microfilaments suggest they play a role in the process of ionophore-induced granule release from polymorphonuclear leukocytes. The ionophore antibiotic A23187, extracted from cultures of Streptomyces chartreusensis, has been shown to alter the permeability of biological membranes to calcium (Reed and Lardy, '72). Such phenomena as lymphocyte mitogenesis (Hovi et al., '76; Luckasen et al., '74), salivary gland secretion (Prince et al., '731, leukocyte chemotaxis (Estensen et al., '76; Wilkinson '751, lymphocyte agglutination (Poste and Nicholson, '76) and capping (Poste and Nicholson, '76; Schreiner and Unanue, '76) have been shown to be initiated, or otherwise affected by the changes in intracellular calcium levels induced by this ionophore. Recent biochemical evidence has indicated that this ionophore enhanced secretion of the specific granule enzyme, lysozyme, from human neutrophils, thus implicating calcium in the induction of its release (Estensen et al., '76; Goldstein et al., '74). In the present report, transmission and scanning electron microscopy were employed to assess the structural changes that occur during granule release in rabbit PMN's exposed to the ionophore A23187.
Scandinavian journal of haematology, 1979
The extracellular release from human neutrophils of the primary (azurophil) granule constituents, myeloperoxidase (MPO), chymotrypsin-like cationic protein (CCP), collagenase and lysozyme, and the secondary (specific) granule constituents, lactoferrin and lysozyme, was measured during ingestion of staphylococcus protein-A-IgG complexes. In buffer, lactoferrin release was consistently higher than that of the other protein. In serum, lactoferrin release increased concomitantly with ingestion, whereas the rate of lysozyme and especially of MPO release were stimulated to a higher degree than ingestion. Magnesium (0.5--2 mM) was more potent than calcium (0.5--2 mM) in promoting release but these cations worked synergistically. Zinc (0.5--4 mM) was found to be a potent and selective inhibitor of collagenase release. Manganese (0.25--4 mM), which inhibited the ingestion of SpA-IgG complexes, also inhibited release of CCP, collagenase, lysozyme and MPO, but actually stimulated lactoferrin r...
Individual and synergistic effects of rabbit granulocyte proteins on Escherichia coli
Journal of Clinical Investigation, 1994
Affinity purification of crude acid extracts of rabbit polymorphonuclear leukocytes using Escherichia coli (J5) as adsorbent yields the bactericidal/permeability-increasing protein (BPI), two 15-kD species (pl5s), and the two most potent (cationic) defensin species (neutrophil peptides [NP] -1 and -2). Tested in buffered isotonic medium, the relative antibacterial potency of these proteins against E. coli J5 is BPI (IC50 0.2 nM) > pl5A (10 nM) > NP -1 (400 nM). Sublethal doses of pl5A or NP-1 can synergize with BPI to decrease the dose required to inhibit the growth of E. coli by up to 50-fold. BPI and pl5A display similar features of antibacterial action distinct from defensin NP-1, but NP-1 acts synergistically only with BPI and not with pl5A. All aspects of the combined action of BPI and NP-1 resemble those observed with higher concentrations of BPI alone, implying that NP-1 enhances BPI potency. Neither NP-1 nor pi5A alter the amount of BPI binding to E. coli but BPI enhances binding of pi5A to E. col, raising the possibility that synergy between these two proteins may occur at least partially at the level of binding. The potent synergistic actions of these proteins can also be demonstrated against serum-resistant clinical isolates of encapsulated E. coli tested in whole blood and plasma ex vivo, suggesting that such combined action may contribute to host defense in vivo.
Journal of Immunological Methods, 1989
The quantitation of CAP57, a highly hydrophobic, native cationic antigen of human polymorphonuclear leukocytes has been achieved using ELISA. An important feature determining the sensitivity and precision of the ELISA was the reduction of non-specific protein-protein binding, particularly in the inhibition assays, thus eliminating high backgrounds obtained with presently available methodology. Washing of the solid phase-bound antigen and blocking of the non-specific binding sites using a potassium phosphate buffer containing heparin largely contributed to this increased sensitivity. The inhibition assays were conducted using antigen concentrations over the range of 0.9-120 ng. The assay is highly specific and can be performed using monoclonal antibodies and polyclonal antibodies. Non-specific reactions were observed only when high concentrations of antigen (> 100 ng) were present in the inhibition mixture. The technique as described is extremely simple, highly reproducible and could be of value in the detection of cationic antimicrobial proteins in the clinical setting in the future.
Biochimica Et Biophysica Acta-molecular Cell Research, 1989
The initiation of the respiratory burst and the degranulation of human polymorphonuclear leukocytes (PMN) in response to stimulation by uropathogenic strains off Escherichia coil is dependent on the expression of Type | fimhriae by those strains. These PMN responses correlate with an increasing tendency of the interacting E. coil strain to be retained on hydrophobic columns. The present work assessed the measurement of relative surface hydrophobicity in relation to PMN activation. Type | fimbr|ate organisms bound most readily to Octyl-Sepharose columns and were strongly agglutinated in the salt aggregation test. |n contrast, the same organisms partitioned to the dextran-rich (hydrophilic) phase of aqueous two-polymer phase systems. Electron microscopic observation of the organisms eluted from the Octyi-Sepharose columns and of the organisms recovered from both phases of the aqueous two-phase systems demonstrated, however, that both Type I and P-fimbriate organisms were retained on the columns and partitioned into the dextran-rlch phase as a consequence of their being fimbriate and failed to identify this as a major factor in the activation of PMN. lh addition electron microscopy demonstrated that e#ch P-fimbriate population had fewer organisms expressing fimbriae than did Type I fimbriate populations, confirming the importance of phase variation as a factor affecting the physicochemical characteristics of a bacterial population.
Infection and Immunity, 1983
In the present study, we assayed protein iodination in human granulocytes after interaction of the cells with mannose-specific (MS) type 1 fimbriated (MS+) and nonfimbriated (MS-) phenotypes of Escherichia coli pretreated with various amounts of anti-E. coli and antifimbrial antibodies. The MS+ phenotype stimulated protein iodination in granulocytes and possessed potent MS activity as measured by yeast aggregometry. In contrast, the MS- phenotype lacked all these activities. MS+ pretreated with moderate concentrations of antibodies, however, showed up to a 15-fold increase in granulocyte stimulation as compared to granulocyte stimulation induced by the non-antibody-treated MS+ phenotype or by the antibody-treated MS- phenotype. This marked antibody-mediated increase in stimulation of granulocytes was (i) dependent on the antibody concentrations, (ii) markedly reduced by methyl-alpha-D-mannoside, (iii) caused by immunoglobulin G as well as by F(ab')2, (iv) caused only by antifimb...
Biochimica Et Biophysica Acta-molecular Cell Research, 1990
The generation of the 5-1ipoxygenase product, leukotriene B 4 (LTB4) by human mononuclear phagocytes (monocytes) following incubation with 25 different uropathogenic strains of Escherichia coli correlated with the haemolytic activity of the strains (r--0.572, P < 0.01). LTB 4 generation by human neutrophils (PMN), however, was unrelated to this haemolytic potential (r= 0.164). In contrast, both prelabelled monocytes and PMN were stimulated by haemolytic strains of E. coli and by haemolytic culture supernatants to release significant amounts of [3Hlarachidonic acid. There was a significant correlation between haemolytic activity and [3H]arachidonic acid release generated by individual strains from monocytes (r--0.804, P < 0.001) and PMN (r--0.888, P < 0.001). In addition, nonhaemolytic strains but not their culture supernatants were capable of causing slow release of both [3H]arachidonic acid and LTB 4 from PMN and mononuclear cells. These results suggest that both the possession of haemolytic activity, and the direct interaction of bacteria with the leukocyte surface are mechanisms by which uropathogenic strains of E. coli may cause the release and metabolism of araehidonic acid. In addition, there was synergistic augmentation by nonhaemolytic bacteria of the PMN LTB 4 response to haemolytic culture supernatants or to low doses of the calcium ionophore A23187. These results support an ionophore-like mechanism for the activation of the cell by haemolysin. LIB 4 generation by PMN incubated with haemolytic supernatants was also augmented by particulate zymosan in a manner dependent on the dose of zymosan, suggesting that the direct interaction of E. coli with PMN may involve an activation mechanism similar to that for zymosan. These results demonstrate differing responses of peripheral mononuclear cells and PMN from the same donors to identical strains of E. coli and suggest that the generation of the potent chemotactic agent LIB 4 in response to E. coli infection in vivo need not depend solely on the elaboration of cytotoxic haemolysins by individual strains.