Thyrotropin-specific binding to human peripheral blood monocytes and polymorphonuclear leukocytes.

[125I]Labeled thyrotropin binding to leukocytes has been studied using lymphocyte-, monocyte- and polymorphonuclear leukocytes-enriched preparations obtained by centrifugation of Ficoll-Angiocontrix gradients and Sephadex G-10 adherence. From the relation between thyrotropin binding and phagocytosis as shown by latex beads ingestion, it is concluded that the hormone binds essentially to monocytes and polymorphonuclear leukocytes. Equilibrium association constants of the high-affinity, low-capacity sites (1 nM-1) are similar to those found in isolated thyroid cells or in thyroid plasma membranes. The role of thyrotropin in the regulation of phagocytosis by leukocytes is discussed.     

Specific binding of B-CLL cell-derived chemokinetic inhibitory factor (CIF) to human polymorphonuclear leukocytes

The chemokinetic inhibitory factor (CIF) is a recently described B-cell derived lymphokine that mediates a chemokinetic inhibitory effect on human polymorphonuclear leukocyte (PMN) migration. In the present report the interaction of CIF with the neutrophil plasma membrane was studied. Normal human peripheral blood neutrophils and purified neutrophil plasma membranes selectively removed biologic activity from CIF-containing concentrates obtained during the purification procedure from conditioned medium. Removal was obtained at both 4 degrees C and 37 degrees C. Furthermore, HL-60 cells treated with dimethyl sulfoxide removed CIF activity (granulocyte-like cells) but HL-60 cells treated with 12-O-tetradecanoylphorbol-13-acetate (macrophage-like cells) did not. Purified human blood monocytes, cells from the macrophage-like U-937 cell line and cells from the basophilic leukemia cell line KU-812 did not remove CIF. These studies suggest that neutrophils express specific binding sites for CIF-activity.     

Antibiotic proteins of human polymorphonuclear leukocytes.

Nine polypeptide peaks with antibiotic activity were resolved from human polymorphonuclear leukocyte azurophil granule membranes. All but 1 of the 12 constituent polypeptides were identified by N-terminal sequence analysis. Near quantitative recovery of protein and activity permitted an assessment of the contribution of each species to the overall respiratory-burst-independent antimicrobial capacity of the cell. Three uncharacterized polypeptides were discovered, including two broad-spectrum antibiotics. One of these, a defensin that we have designated human neutrophil antimicrobial peptide 4, was more potent than previously described defensins but represented less than 1% of the total protein. The other, named azurocidin, was abundant and comparable to bactericidal permeability-increasing factor in its contribution to the killing of Escherichia coli.     

Phagocytosis and killing of Brucella by human polymorphonuclear leukocytes

Although cellular immunity involving activated macrophages is important in resistance to Brucella, serum factors and polymorphonuclear leukocytes (PMNLs) play some role in the initial response to infection. The interaction between human PMNLs and virulent and attenuated strains of Brucella abortus and Brucella melitensis was studied by in vitro techniques. Virulent and attenuated strains of both species were rapidly phagocytosed after opsonization with normal human serum (NHS); nonopsonized bacteria were not phagocytosed. In contrast, NHS devoid of detectable antibodies was bactericidal for strains of B. abortus but not of B. melitensis. In addition, intracellular killing of ingested bacteria was shown for virulent B. abortus but not for B. melitensis. Ultrastructural studies revealed morphological alterations in about one-half of phagocytosed B. abortus and B. melitensis after incubation for 10 min; thereafter, nearly 100% of B. abortus showed some degree of degeneration, whereas B. melitensis remained intact during 120 min of observation.     

Deferoxamine enhances phagocytic function of human polymorphonuclear leukocytes

Inhibition of the iron-mediated generation of toxic oxygen species by polymorphonuclear leukocytes (PMN) might prevent oxidative damage and thus enhance phagocytic function of PMN. To investigate this point, we studied the effect of the specific iron chelator, deferoxamine, on the antibacterial function of PMN. PMN were incubated for 20 hr with various concentrations of deferoxamine at 37 degrees C in medium containing 0.54 microM endogenous iron. The cells were then washed, and the phagocytic cell function was assessed. The results were compared with those for control PMN preincubated for 20 hr without deferoxamine, and those of nonincubated PMN. Compared with that of control PMN, the uptake of radiolabeled Staphylococcus aureus by PMN treated with 1 microM-1 mM deferoxamine was, on average, 10%-20% higher. This effect was not observed when iron-saturated deferoxamine (DFO) was used. Bacterial uptake was similarly increased in nonpreincubated PMN or PMN preincubated for 20 hr at 4 degrees C instead of 37 degrees C. The intracellular killing capacity of both deferoxamine-treated and control PMN exceeded 90%. PMN incubated for 20 hr at 37 degrees C with DFO not only phagocytosed more bacteria than control cells, but were also capable of killing the greater number of bacteria ingested. This increased activity of deferoxamine-treated PMN was accompanied by enhanced generation of chemiluminescence and production of superoxide during phagocytosis of S. aureus. These findings indicate that deferoxamine may enhance the antibacterial activity of PMN by protecting the cells against damage by iron-mediated generation of toxic oxygen metabolites in resting PMN.     

Doxycycline effects on the adherence of polymorphonuclear leukocytes to an albumin-coated glass surface

The effect of doxycycline on polymorphonuclear leukocyte (PMN) adherence to albumin-coated glass surfaces was studied in the absence and presence of the chemotactic peptide FMLP. Three concentrations of doxycycline were studied, one subtherapeutic (0.1 micrograms/ml), one therapeutic (1.0 micrograms/ml) and one supertherapeutic (10 micrograms/ml). PMN adherence was maximal after incubation for 5 min. FMLP did not affect PMN adherence in the present assay system and at the concentration studied (10(-7)M). PMN adherence remained stable and unaffected in the tested doxycycline concentrations. Thus, the present study could not confirm the reported and challenged doxycycline inhibition of PMN adherence.     

Functional analysis of the marginating pool of human polymorphonuclear leukocytes

The intravascular pool of human polymorphonuclear leukocytes (PMN) is composed of one compartment which is circulating and another that is marginated to the vascular endothelium. Administration of B-adrenergic agonists leads to a rapid demargination with an increase in the circulating PMN pool. The marginating PMN has previously been stated to represent an older PMN based on a higher cytochemical alkaline phosphatase activity. With the understanding that circulating PMN are heterogeneous with respect to function and size we undertook the present study to evaluate the contribution of the marginating PMN to functional and volume-dependent heterogeneity. We found that PMN isolated 7 min after epinephrine administration, presumably enriched by marginating PMN, were not different in volume, biochemically measured alkaline phosphatase activity, stimulated superoxide anion release, degranulation, or phagocytosis. These data suggest that the circulating and marginating pools of PMN are interchangeable and that the marginating PMN are not enriched by a particular subpopulation of PMN.     

The interaction between Treponema pallidum and human polymorphonuclear leukocytes

The interaction between polymorphonuclear leukocytes (PMNLs) and Treponema pallidum was studied. Intradermal injection of greater than or equal to 10(6) T. pallidum into rabbits caused a rapid accumulation of PMNLs. Human serum released chemotaxigenic factor (C5a) during incubation in vitro with T. pallidum. Incubation of T. pallidum with human PMNLs in vitro (ratio, 100:1) stimulated chemiluminescence. These responses were dependent upon the presence of both antibody and complement and were greatest when serum from a patient with late secondary syphilis was used as a chemotaxigenic source or for opsonization. Electron microscopic studies documented the rapid uptake of T. pallidum into membrane-bound vacuoles in the human PMNLs in vitro after incubation for as little as 5 min, with leukocyte degranulation and loss of treponemal integrity observed after 4 hr. T. pallidum were found within PMNLs 3 hr after intradermal inoculation of rabbits. These data show that PMNLs are attracted to, and appear to ingest, T. pallidum, but they fail to explain why inoculation of these organisms is not followed by eradication.     

A quantitative colorimetric method to evaluate the functional state of human polymorphonuclear leukocytes

Summary The colorimetric assay previously described by Mosmann [11] for the measurement of cell viability and proliferation has been modified for the assessment of the functional state of human polymorphnuclear cells (PMNs). The ability of PMNs to reduce the tetrazolium salt MTT to formazan reflects directly the degree of stimulation induced by various agents. The underlying mechanism of MTT-reduction to formazan seems to be similar to that of nitroblue tetrazolium (NBT)-reduction. In contrast to the NBT-reduction assay, the formazan produced from MTT can easily be measured by an ELISA reader. Parallel experiments revealed a qualitative correlation between the concentration of formazan produced from MTT and the concentration of cytochrome C reduced by PMNs. Although oxidative burst may not be the actual lytic mechanism in cellular cytotoxicity of PMN, we also observed an association between MTT-reduction capacity and the cytotoxic activity of PMNs from normal donors in antibody dependent cellular cytotoxicity. Our results indicate that the MTT-reduction assay can be employed to estimate the functional state of polymorphnuclear granulocytes.Supported in part by grant no. CA33484 from the NIH     

Hyposmolarity stimulates myeloperoxidase exocytosis from human polymorphonuclear leukocytes

Medium hyposmolarity induced in human polymorphonuclear leukocytes treated with cytochalasin B a rapid exocytosis of the lysosomal enzyme, myeloperoxidase (MPO), which was linearly proportional to the degree of hyposmolarity between a 5 and 30% decrease (r = 0.92, p less than 0.001). Cell viability was unaffected by the hyposmolarity. The kinetics of MPO exocytosis induced by opsonized zymosan (OZ) and hyposmolarity were indistinguishable; the combination of hyposmolarity and OZ was additive. Since hyposmolarity similarly stimulates a burst of hormone secretion by perifused adenohypophyseal and pancreatic islet cells, the authors suggest that hyposmolarity-induced exocytosis is a general cellular phenomenon.     

Platelet-leukocyte interaction: selective binding of thrombin- stimulated platelets to human monocytes, polymorphonuclear leukocytes, and related cell lines

The association of platelets with leukocytes was investigated, using gel-filtered platelets stimulated with thrombin and then fixed with formaldehyde. Evidence is presented that stimulation of gel-filtered platelets with low concentrations of thrombin (0.01 to 0.1 U/mL) induces the expression of surface determinants interacting strongly with monocytes and polymorphonuclear leukocytes (PMNs) but only weakly with lymphocytes. Both monocyte-platelet binding and PMN-platelet binding occurred at 37 degrees C and more intensively at 0 degrees C; it was Ca2+-dependent and was unaffected by the addition of sodium azide. The binding also occurred with the monocytoid cell lines HL 60 and U 937 in exponential growth and was much less two days after induction of terminal differentiation by phorbol myristate acetate (PMA). No other tested cell lines (B cells, T cells, and myeloid cells) bound thrombin-stimulated platelets. Monocyte-derived macrophages kept in culture for one week also exhibited reduced binding of thrombin- stimulated platelets. IgG and fibronectin could be ruled out as ligands that mediate binding.     

Arachidonic acid metabolism and the adhesion of human polymorphonuclear leukocytes to cultured vascular endothelial cells

Polymorphonuclear leukocytes (PMN) adhere to the vascular endothelial lining in vivo and to the surfaces of cultured endothelial cells in vitro, but the mechanisms of these cellular interactions remain unclear. Arachidonic acid metabolites, both cyclooxygenase- and lipoxygenase-derived, have been shown to influence PMN locomotion, secretion, and adhesion to artificial surfaces. To determine whether such mediators also are involved in regulating PMN-endothelial cell interactions, we have examined the effects of prostacyclin and various inhibitors of arachidonic acid metabolism on the adherence of radiolabeled PMN to cultured bovine aortic endothelial cells. Confluent endothelial monolayers were incubated with washed suspensions of radiolabeled human PMN (which contained less than 1% platelet contamination) at 37 degrees C for 30 min, then subjected to a standardized wash procedure and the number of adherent leukocytes determined radiometrically. Under basal conditions, i.e., in the absence of exogenous activating stimuli, 4,163 +/- 545 PMN adhered per square millimeter of endothelial surface (mean +/- SEM, n = 12). This basal adhesion (which corresponds to approximately 4-5 leukocytes per endothelial cell) was unaffected when the leukocytes and endothelial monolayers were pretreated with cyclooxygenase inhibitors (100 microM aspirin or 1-5 microM indomethacin) or PGI2 (10(-9)-10(6) M). Thus, basal PMN-endothelial adhesion in this in vitro model system does not appear to be dependent on endogenous cyclooxygenase derivatives of arachidonate or to be sensitive to inhibition by exogenous prostacyclin. In contrast, leukocyte adhesion was significantly reduced by pretreatment with 5,8,11,14- or 4,7,10,13-eicosatetraynoic acid, 0.5- 5 mM sodium salicylate, or 10-1,000 microM indomethacin, antiinflammatory agents that can interfere with the metabolism of arachidonic acid via non-cyclooxygenase-dependent mechanisms. These observations may be relevant to the interactions of circulating PMN with vascular endothelium under both physiologic and pathophysiologic conditions in vivo.     

Release of tumor necrosis factor by human polymorphonuclear leukocytes

Evidence is presented that human polymorphonuclear neutrophils (PMN) can be induced to produce tumor necrosis factor (TNF). Other investigators have previously reported that TNF has been induced from macrophages by bacteria and, more recently, from natural killer cells by certain tumor cells. Our laboratory has reported that the opportunistic fungi, Candida albicans, can induce TNF, not only from human monocytes, but also from Percoll-fractionated large granular lymphocytes. We now report that incubation of PMN with C albicans for 3 hours was sufficient for detection of TNF release, and peak induction was observed at 8 to 18 hours. This release was inhibitable by actinomycin D, an inhibitor of RNA synthesis, as well as by emetine and cycloheximide, which block protein synthesis. The TNF produced by PMN was neutralized by specific monoclonal antibodies against human TNF. These results represent an important finding that TNF production is a normal response of PMN to stimulation by fungi such as C albicans and suggest that the release of TNF may be related to autocrine activation of PMN effector function to control Candida growth.     

Vero cytotoxin binding to polymorphonuclear leukocytes among households with children with hemolytic uremic syndrome

Hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in childhood, can be caused by different serotypes of vero cytotoxin (VT; i.e., Shiga toxin)-producing Escherichia coli (VTEC). Recently, VT was shown to bind to polymorphonuclear leukocytes (PMNL) in the systemic circulation of patients with HUS. This study investigated whether VT bound to PMNL could be detected in persons in households with patients with HUS. Serum antibodies against E. coli O157 and, when available, fecal samples from patients with HUS and household members were studied for the presence of VTEC infection. The circulating PMNL of 82% of the household members were positive for VT, whereas stool and/or serum examination showed only 21% positivity. Thus, current methods underestimate the number of infected persons in households with patients with HUS.     

Metabolic, membrane, and functional responses of human polymorphonuclear leukocytes to platelet-activating factor

The phospholipid mediator of anaphylaxis, platelet-activating factor (PAF) is chemotactic for polymorphonuclear leukocytes (PMN). We have examined this agent's effects on several other PMN functions. Human PMN were prepared from heparinized venous blood by Ficoll gradient. Metabolic burst was examined by measurement of O2 use and O2.- production in the presence or absence of PAF (10(-6)--10(-9) M). Unless cells were treated with cytochalasin-B (5 micrograms/ml), no significant respiratory burst was demonstrated. However, pretreatment with PAF (10(-7) M) enhanced approximately threefold the O2 utilization found when cells were subsequently stimulated with 10(-7) M FMLP. PAF also stimulated arachidonic acid metabolism in 14C-arachidonic acid- labeled PMN. Thin-layer chromatography analysis of chloroform-methanol extracts showed substances that comigrated with authentic 5- hydroxyeicosatetraenoic acid had a marked increase in radioactivity following PAF stimulation at 10(-7) M. PAF failed to stimulate release of granule enzymes, B-glucuronidase, lysozyme, or myeloperoxidase unless cytochalasin-B were added. PAF from 10(-6) M to 10(-10) M affected PMN surface responses. PMN labeled with the fluorescent dye, chlorotetracycline, showed decreased fluorescence upon addition of PAF, suggesting translocation of membrane-bound cations. Further, the rate of migration of PMN in an electric field was decreased following PAF exposure, a change consistent with reduced cell surface charge. PMN self-aggregation and adherence to endothelial cells were both influenced by PAF (10(-6) M--10(-9) M). Aggregation was markedly stimulated by the compound, and the percent PMN adhering to endothelial cell monolayers increased almost twofold in the presence of 10(-8) M PAF. Thus, PAF promotes a variety of PMN responses: enhances respiratory burst, stimulates arachidonic acid turnover, alters cell membrane cation content and surface charge, and promotes PMN self- aggregation as well as adherence to endothelial cells.     

Action of collagenase and elastase from human polymorphonuclear leukocytes on human articular cartilage

Summary Collagenase from human polymorphonuclear leukocytes (neutrophil collagenase) attacks collagen type II in solution at a rate intermediate to those of type I and III collagens. This enzyme alone is not able to initiate degradation of native human articular cartilage. If the cartilage is first treated with leukocyte elastase, collagenase slowly degrades collagen.Confirming earlier findings by other investigators, elastase has a dual action on cartilage: The enzyme removes proteoglycans, thus demasking collagen fibers and giving collagenase access to them, and solubilizes collagen at a sizable rate.Although neutrophil collagenase cleaves collagen type II in solution at a high rate, the native, cross-linked status of collagen in cartilage makes it a relatively poor substrate for this enzyme. On a weight by weight scale, elastase and collagenase display about the same collagenolytic potential on human articular cartilage.The elastase/collagenase system from human polymorphonuclear leukocytes could represent a cooperative proteolytic complex in the destruction of cartilage in rheumatoid arthritis.     

Diminished release of lactoferrin from polymorphonuclear leukocytes of human neonates

In neonatal and adult polymorphonuclear leukocytes (PMN) we determined the content and the release of beta-glucuronidase, myeloperoxidase, lysozyme and lactoferrin. We found an equal total content of these proteins in adult and neonatal PMN, except for a lower lysozyme concentration in neonatal PMN. In the presence of opsonized zymosan myeloperoxidase, lysozyme and beta-glucuronidase were released in equal amounts; lactoferrin, however, was released to a lower rate from neonatal than from adult PMN (p less than 0.0005).