Characterization of the human interleukin 1 receptor on human polymorphonuclear leukocytes

Interleukin 1 (IL-1) is a mediator of inflammation with multiple proinflammatory and immunologic enhancing activities. Human polymorphonuclear leukocytes (PMN) also play a major role in the inflammatory response. We have found that PMN possess a single type of high affinity receptor for human recombinant (r) IL-1 alpha with an apparent dissociation constant of 0.28 nM. Approximately 700 receptors are present per cell. Binding is rapid with 50% of maximal binding occurring within 20 min at 4 degrees C. Internalization of the receptor occurs within 25 min after shifting the cells to 37 degrees C. The receptor exhibits an apparent molecular weight of approximately 60-70 kDa. Electron microscopic autoradiography studies reveal that the 125I-rIL-1 alpha localized in the nucleus within 180 min after shifting cells to 37 degrees C. The accumulation of relatively high levels of 125I-rIL-1 alpha in the nucleus is consistent with earlier observations on the nuclear localization of IL-1 in T lymphocytes. The possibility that IL-1 may exert a direct action in the nucleus remains to be determined.     

Characterization of effect ofN-formyl-methionyl-leucyl-phenylalanine on leukotriene synthesis in human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNLs) were exposed toN-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) in the presence or absence of exogenous arachidonic acid. Analysis of incubation mixtures by high-performance liquid chromatography showed that f-Met-Leu-Phe stimulated the synthesis of 5-hydroxy-eicosatetraenoic acid (HETE) and of leukotriene B₄ (LTB₄) which was rapidly metabolized into 20-OH-LTB₄ and 20-COOH-LTB₄. The stimulatory effect of f-Met-Leu-Phe was dose and time dependent. The tripeptide showed maximum stimulatory activity at the concentration of 1 M and after 20–30 min of incubation. Addition of arachidonic acid to the f-Met-Leu-Phe-stimulated PMNLs resulted in an increase in the synthesis of LTB₄ and 5-HETE. Pretreatment of the PMNLs with cytochalasin B strongly potentiated (up to six-fold) the stimulatory effect of f-Met-Leu-Phe on 5-lipoxygenase product synthesis, whereas cytochalasin B alone or with arachidonic acid had no significant effect. The tripeptide did not increase the synthesis of plateletderived 12-HETE, and 12-hydroxyheptadecatrienoic acid, or of PMNL-derived 15-HETE, suggesting that its action was selective for PMNL 5-lipoxygenase. The present data indicate that f-Met-Leu-Phe causes the release of arachidonic acid from cellular lipids and activates the 5-lipoxygenase.     

Intercellular junction formation between polymorphonuclear leukocytes, and between polymorphonuclear leukocytes and tumor cells

Morphologic evidence of intercellular junctions was found between polymorphonuclear leukocytes (PMNs) and between PMNs and tumor cells. All these junctions displayed electron-dense plaques along the plasma membranes with linear spaces in between. These plaques were finely granular and devoid of filaments. Fine granules were also observed in some of these linear spaces. The significance of these morphologic findings is not known but may be elucidated by a correlative type of study.     

Electron microscopic and autoradiographic study of the interaction of human polymorphonuclear leukocytes and staphylococci

Leukocyte suspensions of normal subjects and patients with burns and wounds were incubated with Staphylococcus epidermidis and incorporation of 3H-uridine into leukocytes and bacteria was examined by electron microscopic autoradiography. As the maturation of the precursor cells of polymorphonuclear leukocytes (PMNL) progressed the level of RNA synthesis in them was found to decrease gradually and in PMNL RNA synthesis ceased completely. If PMNL take part in phagocytosis, however, RNA synthesis is found in a considerable number of them. The results indicate that RNA synthesis in PMNL is associated with the formation and functional condition of primary and secondary granules. Certain conditions affecting the viability of staphylococci ingested by phagocytes are described.     

Effects of N-acetylcysteine on human polymorphonuclear leukocytes

N-acetylcysteine (NAC) at concentrations from 0.39 micrograms/ml to 100 micrograms/ml did not affect chemotaxis under agarose of human polymorphonuclear leukocytes (PMNLs). No reduction of phagocytic or bactericidal capacity was found in PMNLs exposed to NAC at the same concentrations. At high concentrations of NAC (25-100 micrograms/ml) a distinct inhibition of the chemiluminescent response to formylmetionyl-leucyl-phenylalanine (fMLP) known to be associated with mainly extracellular metabolic processes, was observed, consistent with the well known scavenger effects of the drug. The response to opsonized zymosan, which reflects mainly intracellular metabolic activity, was less marked. At a still higher concentration of NAC (500 micrograms/ml), a distinct effect on both intra- and extracellularly generated chemiluminescence could be demonstrated. The lack of inhibitory effects on phagocytosis and intracellular killing in spite of the effects on chemiluminescence indicates that NAC has no negative influence on the antimicrobial activity of PMNLs.     

Characterization of C3a receptor-proteins on guinea pig platelets and human polymorphonuclear leukocytes

The expression of specific membrane receptors for C3a was determined on guinea pig C3a-sensitive (gp R+) platelets and human polymorphonuclear leukocytes (hu PMNL). Binding studies with 125I-labeled C3a from gp or hu sources and Scatchard analysis applied to the binding data revealed the existence of two receptor classes on gp R+ platelets; a high-affinity class with about 200 binding sites/cell and Kd = 1.7 x 10(-9) M, and a relatively low-affinity class with Kd = 10(-8) M and about 500 sites/cell. Hu PMNL express a homogeneous receptor class with Kd = 3 x 10(-8) M and 40,000 sites/cell. Molecular characterization of the C3a receptor on gp R+ platelets was achieved by (a) cross-linking photoaffinity-labeled receptors to bound 125I-labeled C3a; (b) photoaffinity labeling receptors with a 13-amino acid residue C3a analogue 125I-Nap-Ahx-13; and (c) use of chemical cross-linkers like disuccinimidylsuberate to cross-link receptors with 125I-C3a. All three techniques gave rise to very similar labeling patterns. With the photoaffinity labeling methods, a diffuse band pattern was observed with an apparent molecular mass of 95-123 kDa with 125I-C3a as label, and 85-105 kDa with 125I-Nap-Ahx-13 as label. Chemical cross-linking of 125I-C3a revealed three distinct bands with molecular masses of approximately 123, 108 and 95 kDa. Subtracting the contribution of the cross-linked ligands, the C3a receptor on gp R+ platelets appears to be a protein complex, consisting of one to three components with estimated molecular masses between 83-114 kDa.     

Characterization of lipoteichoic acid binding to polymorphonuclear leukocytes of human blood.

Human polymorphonuclear leukocytes (PMN) were shown to possess specific binding sites for lipoteichoic acid (LTA). LTA binding was reversible and time and temperature dependent. Scatchard plot analysis revealed an apparently single population of 6.6 X 10(6) LTA binding sites per PMN with a dissociation constant of 5.6 microM. Attachment of an avirulent, unencapsulated, M-negative strain of group A streptococci to PMN was inhibited by LTA, but not by other bacterial somatic antigens tested. Occupation of 30% of the LTA binding sites resulted in greater than 70% inhibition of streptococcal attachment to PMN. In contrast, LTA failed to block attachment of Escherichia coli or antibody-coated streptococci, indicating that binding sites for E. coli and the Fc portion of immunoglobulin G are distinct from those for LTA. Immunofluorescent studies demonstrated that LTA remained uniformly bound to PMN membranes for as long as 2 h at 37 degrees C. Cross-linking of PMN-bound LTA with anti-LTA resulted in rapid capping of LTA receptor sites. The results suggest that LTA is a monovalent ligand interacting with mobile receptors in the plasma membrane of PMN.     

Characterization of the IgA receptor from human polymorphonuclear leucocytes.

Human polymorphonuclear leucocytes (PMNs) will phagocytose yeasts opsonized with specific affinity-purified human serum IgA. PMNs also bind to Sepharose beads coated with IgA or IgG, but not to beads coated with bovine serum albumin (BSA) or horseradish peroxidase (HRP). Binding to IgA-Sepharose stimulates the cells to release lysozyme. Affinity chromatography of 125I-labelled PMN membrane proteins on IgA-Sepharose results in isolation of a polypeptide of apparent 60,000 MW. The protein, which is not bound to IgG-Sepharose under the same conditions, appears as a diffuse band on SDS-PAGE, suggesting it is heavily glycosylated.     

Interaction between Chlamydia spp. and human polymorphonuclear leukocytes in vitro

Chlamydia psittaci and Chlamydia trachomatis elementary bodies (EB) incubated in the presence of complement or specific antibody or both caused chemotaxis of human polymorphonuclear leukocytes (PMN) in vitro. Reticulate bodies and culture supernatants had no effect on these cells. The ability of chlamydiae to enter and survive in PMN under nonopsonizing conditions was investigated by measuring the association of 3H-labeled EB and of inclusion-forming units with these phagocytes. Both assays indicated that C. psittaci as well as C. trachomatis EB are efficiently internalized. The mechanism by which this is accomplished is distinct from classical phagocytosis in that it is not dependent upon the presence of complement or antibody. Furthermore, uptake of at least C. psittaci appeared to be rapid, with no additional increase occurring after 15 min. The majority of cell-associated chlamydiae were rendered acid soluble or noninfectious within 1 h. Subsequently, there was a small but steady loss of infectivity for up to 10 h, which may have been due to the conversion of EB to the noninfectious reticulate-body form of the organism. However, even at 10 h after entry a small percentage of bacteria was still capable of infecting a second target cell. This is noteworthy in that PMN are relatively short-lived cells, and after lysis, intracellular organisms may be free to infect adjacent tissue. Electron microscopic observations were consistent with the data on uptake and persistence. The ability of a small percentage of infecting chlamydiae to maintain infectivity in PMN for at least several hours may enable these organisms subsequently to establish productive infection in permissive host cells.     

Synergistic antifungal effect of fluconazole and human polymorphonuclear leukocytes on Paracoccidioides brasiliensis: effect of interferon-gamma and granulocyte-macrophage colony-stimulating factor.

To better understand the in vivo efficacy of fluconazole (FCZ), we investigated the possible synergy of fungistatic FCZ with human polymorphonuclear leukocytes (PMN) against Paracoccidioides brasiliensis (Pb). The effect of interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in this system was also studied. For this purpose, FCZ, PMN, PMN + FCZ, PMN + IFN-gamma, PMN + IFN-gamma + FCZ, PMN + GM-CSF and PMN + GM-CSF + FCZ were co-cultured with Pb and the cfu of Pb was measured. The antifungal effect of FCZ on yeast cells of Pb was concentration-dependent. At 0.1 microg ml(-1), FCZ had no effect on the growth of Pb. At 0.2 microg ml(-1) FCZ showed a growth-inhibitory effect on three isolates of Pb in a long-term (120 h) assay, and at 0.6 microg ml(-1) or higher FCZ was fungicidal. Fungistatic concentration of FCZ (0.4 microg ml(-1)) acted synergistically with fungistatic PMN for killing isolate Bt-4 during the first 24 h of co-culture. Moreover, IFN-gamma and GM-CSF substantially enhanced the synergistic antifungal effect of PMN and FCZ. These findings provide a better understanding of why FCZ is more efficacious in in vivo models of paracoccidioidomycosis than is predicted by in vitro susceptibility tests.     

Release of prostaglandins from human polymorphonuclear leukocytes

Human PMNs release prostaglandins E and F to the surrounding medium when these cells are exposed to zymosan. PGE₁ is the prostaglandin compound found in highest concentration in the medium, and the PGE/PGF balance is approximately 3∶1. Release of prostaglandins is not due to platelet contamination. Agents which inhibit prostaglandin synthesis (indomethacin, aspirin) prevent release of prostaglandins from phagocytic cells. Addition to cells of dibutyryl cyclic 3′,5′-adenosine monophosphate produces striking increases in concentrations of prostaglandins released during ingestion of zymosan. Prostaglandins appear to be synthesized by human PMN during phagocytosis, and their release from cells may help regulate the inflammatory response.     

Oxidation of glucosamine by human polymorphonuclear leukocytes

When exposed to a phagocytic stimulus (opsonized zymosan), human polymorphonuclear leukocytes (PMNs) produced¹⁴CO₂ from [1-¹⁴C]glucosamine at a rate 10–25% of that produced from glucose under the same conditions. The production of CO₂ from glucosamine by intact PMNs was inhibited by glucose and dependent upon activation of the hexosemonophosphate shunt (HMPS). However, the metabolic pathways for the oxidation of glucose and glucosamine by PMNs are not identical. This is suggested by the fact that glucose-6-phosphate dehydrogenase, the initiating enzyme for the HMPS, did not utilize glucosamine-6-phosphate as a substrate. In addition, glucosamine was not oxidized by sonically disrupted PMNs whereas oxidation of glucose by the same preparation was increased sevenfold over intact cells. Taken together, the data suggest that PMNs oxidize glucosamine by converting it to a compound compatible with the enzymes of the HMPS. This conversion requires intact PMNs and/or an as yet unidentified cofactor.     

Effects of pneumolysin on human polymorphonuclear leukocytes and platelets.

Pneumolysin was bound by human polymorphonuclear leukocytes in a reaction which occurred very rapidly at 0 degrees C. Low concentrations of pneumolysin were found to stimulate leukocyte migration and lysosomal enzyme secretion. At increasing lysin levels, inhibition of spontaneous migration and chemotaxis, cell death, and lysis were observed. Pneumolysin was also found to lyse platelets and to activate serum to become chemotactic.     

Antibiotic-induced modification ofBacteroides fragilis and its susceptibility to phagocytosis by human polymorphonuclear leukocytes

Bacteroides fragilis grown in the presence of sub-inhibitory concentrations of clindamycin was shown to be altered its degree of encapsulation and susceptibility to phagocytosis by human polymorphonuclear leukocytes. Little polysaccharide capsule could be demonstrated either by light or transmission electron microscopy when the bacteria were grown anaerobically for four hours in the presence of 1/2 MIC of clindamycin. Such clindamycin-grown cells could be opsonized by normal human serum, and although less complement was consumed in the process, were more effectively taken up by the leukocytes than bacteria grown in the absence of the drug (45 % versus 24 %). It was also shown that drug treatment caused significant cellular leakage in the presence of serum, the³H-label appearing extracellularly. In addition there was greater loss of viability of the bacterial cells grown in the presence of the drug and subsequently exposed to the leukocytes for 60 min.     

Betamethasone modulates the biological function of human polymorphonuclear leukocytes

We have examined the influence of betamethasone (BT) on cyclic AMP (cAMP) metabolism and lysosomal enzyme release from highly purified (approximately equal to 99%) human polymorphonuclear leukocytes (PMNs). Preincubation (1-24 h) of human PMNs with BT (10(-9)-10(-5) M) had no effect on either cAMP content or on beta-glucuronidase release induced by formyl-containing tripeptide (f-met peptide). Preincubation (16-24 h) of PMNs with BT (10(-8)-10(-7) M) dose-dependently potentiated the cAMP accumulation caused by beta-agonists (isoproterenol), adenosine A2/Ra agonist (NECA), prostaglandin E1 (PGE1) and histamine in PMNs. Similarly, BT potentiated the inhibition of f-met peptide-induced beta-glucuronidase release from human PMNs caused by PGE1 (10(-6) M), histamine (2 X 10(-5) M), NECA (10(-4) M) and isoproterenol (10(-6) M).     

Characterization of the interaction of human plasmin with its specific receptor on a group A streptococcus

Certain Group A beta-hemolytic streptococci express a receptor that is capable of specifically binding the human plasma protease plasmin. Once bound, plasmin remains enzymatically active and is unregulated by its naturally occurring inhibitor alpha-2-antiplasmin (Lottenberg, R., C. C. Broder and M. D. P. Boyle, 1987. Infect. Immun. 55: 1914-1918). In this study certain characteristics of the interaction between plasmin and the receptor expressed on a group A beta-hemolytic streptococcus, strain 64/14, were examined. Binding occurred optimally at physiologic pH and ionic strength. The KD was 5 x 10(-11) M and there were approximately 800 receptors per bacterium. Mouse passage of strain 64 had no significant effect on the KD of the receptor. Binding of plasmin to the bacteria was inhibited by lysine and epsilon-aminocaproic acid in a concentration dependent manner. Similarly these amino acids would displace pre-bound plasmin from the bacteria. These findings suggest a role for plasmin's high affinity lysine binding site in the interaction of plasmin with the bacteria.     

Histamine H-1 binding site on human polymorphonuclear leukocytes

Neutrophilic polymorphonuclear leukocytes (PMN) infiltrate the sites of allergic reactions and may respond to histamine, one of the major mediators of allergy. In order to characterize histamine interactions with PMN, the binding of [³H]pyrilamine was studied. Human PMNs bind [³H]pyrilamine in a specific, saturable, and reversible fashion and demonstrate specificity (H-1 antagonists > histamine > H-2 antagonists) for the competitive binding agents studied. Human PMNs have a homogeneous population of H-1 receptors of moderate affinity (K _d=52 nM) in large number (265 × 10³/cell) which do not demonstrate cooperativity. Thus PMNs attracted to sites of allergic inflammation have H-1 binding sites which may respond to histamine stimulation.     

Purification and characterization of calpain from human polymorphonuclear leukocytes

Recent studies have demonstrated that a calcium-sensitive protease converts Ca²⁺/phospholipid-dependent protein kinase C to a Ca²⁺/phospho-lipid-independent form during the activation of human neutrophils. In this paper, the results of the purification and characterization of a calcium-dependent cytosolic protease from neutrophils is reported. Calcium-dependent protease has been purified 1062-fold from human neutrophils and behaves as a single species on native polyacrylamide gels. The protease is active in the neutral pH range with no observable activity at pH values greater than 8.0, has an absolute requirement for calcium for expression of activity with half-maximal activity observed at 12 /M free calcium, and has an apparent molecular weight of 110,000 based on gel filtration. The protease requires the presence of dithiothreitol for activity and is inhibited by sulfhydryl inhibitors, leupeptin, and antipain but not by serine protease inhibitors, pepstatin, or orthophenanthroline. The protease is also susceptible to inactivation by autoproteolysis. Based on the similarities of this calcium-dependent protease with calpains from a variety of other mammalian tissues, the protease isolated from human neutrophils appears to be a calpain I.     

Chemiluminescence spectra of human myeloperoxidase and polymorphonuclear leukocytes.

The light emission spectra of myeloperoxidase-H2O2-Cl- and phagocytizing polymorphonuclear leukocytes were estimated by a computer simulation technique by using light transmittance data from nine band-pass filters. No shift in the chemiluminescence spectrum of either system was observed during the course of the reactions, suggesting that the light-generating mechanisms remain constant after their initiation. Transmittances were virtually identical for both myeloperoxidase-H2-O2-Cl- and polymorphonuclear leukocyte reactions, suggesting that the light-generating mechanisms are identical and leading to the estimation of nearly identical spectra. Both spectra were broad, with maximum light emission near 570 nm.