Normal PMN Research Articles

Immune complex-stimulated neutrophil LTB4 production is dependent on beta 2 integrins.

The beta 2 integrins (LFA-1, Mac-1, and p150,95) are critical for many adhesive functions of leukocytes. Although the binding of the IgG-opsonized particles occurs normally in the absence of beta 2 integrins, phagocytosis of IgG-opsonized particles by activated neutrophils (PMN) requires these integrins. This observation suggests a role for beta 2 integrins in phagocytosis subsequent to particle binding. To investigate the mechanism of involvement of beta 2 integrins in IgG-mediated functions, we examined the role of beta 2 integrins in adhesion to immune complex (IC)-coated surfaces. Initial adhesion and spreading on IC-coated surfaces were equivalent in control and beta 2-deficient phagocytes. However, both genetically beta 2-deficient PMN and PMN treated with the anti-beta 2 mAb IB4 subsequently detached from the IC-coated surfaces. To determine whether biochemical consequences of IgG activation were also affected by beta 2 deficiency, LTB4 production in response to Fc receptor ligation was assessed. LTB4 production by beta 2-deficient PMN adherent to IC-coated surfaces was markedly decreased in comparison with control PMN. Importantly, LTB4 production by PMN stimulated with fluid phase heat-aggregated IgG also required the beta 2 integrins, showing that the defect was not a simple consequence of abnormal adhesion. In contrast, superoxide production by IC-adherent PMN was equivalent in control and beta 2-deficient PMN. The initial rises in intracytoplasmic [Ca2+]i in response to aggregated IgG also were unaffected by inhibition of beta 2 integrins. These data show that lack of beta 2 integrins does not inhibit all FcR-dependent signal transduction. Finally, LTB4 production by normal PMN adherent to ICs was inhibited by antibodies to FcRII, but not FcRIII, showing that FcRII ligation was required for this effect. Together these data identify a role for the beta 2 integrins in a signal transduction pathway leading to sustained adhesion and LTB4 production in response to IC. Since both beta 2 integrins and FcRII are required for these effects, the data further suggest cooperation between these receptors in generating PMN activation in response to IC stimulation.

Activation of tumor necrosis factor-alpha production from human neutrophils by IL-2 via IL-2-R beta.

In addition to T cells, NK cells, B cells, and monocytes, we provide new evidence that human polymorphonuclear neutrophils (PMN) can be functionally activated by IL-2 via binding to IL-2R beta expressed on the cell surface. Brief exposure of normal PMN to human rIL-2 enhanced both transcriptional and translational expression of TNF-alpha. The release of TNF-alpha protein by IL-2-treated PMN was inhibitable by a specific mAb against human IL-2-R beta. The response to IL-2 was dose and time dependent with the increase in TNF-alpha mRNA detected maximally 3 h after IL-2 exposure, followed by a continuous maintenance of high mRNA levels up to 18 h. The TNF-alpha mRNA was significantly increased above the medium control level, with as little as 10 U/ml of IL-2. Maximal transcription was obtained with 1000 U/ml of IL-2, which achieved the level observed with known neutrophil activating factors such as granulocyte-macrophage-CSF, IL-8, and Candida albicans. Using actinomycin D, it was found that new and continuous synthesis of a labile TNF-alpha mRNA was responsible for the observed high levels of transcripts. Of significance was the observation that cycloheximide could selectively modulate TNF-alpha mRNA transcription in neutrophils, depending on the cytokine used. Cycloheximide did not affect or alter TNF-alpha mRNA induction in IL-2-treated neutrophils but abrogated it in granulocyte-macrophage-CSF-treated neutrophils and superinduced transcription in C. albicans-treated neutrophils. Thus various control elements must be involved in the transcription of the TNF-alpha genes that are responsive to different cytokines and activating factors. The induction of TNF-alpha and functional activation of neutrophils by IL-2 is therefore an important immunomodulatory property of IL-2 that has not heretofore been recognized.

Function associated with IL-2 receptor-beta on human neutrophils. Mechanism of activation of antifungal activity against Candida albicans by IL-2.

Polymorphonuclear neutrophils (PMN) are essential components of the host defense system against a wide variety of pathogens. We report here the novel finding that freshly isolated human PMN constitutively express detectable surface levels of IL-2R beta, but not IL-2R alpha, as analyzed by flow cytometry. Northern blot analysis confirmed the constitutive expression of mRNA for IL-2R beta in PMN. Scatchard analysis using 125I-labeled IL-2 demonstrated the presence of approximately 600 intermediate binding IL-2R per PMN, with a dissociation constant of 1.1 x 10(-9) M, similar to that of IL-2 binding to YT-1 tumor cells that specifically express IL-2R beta. More importantly, PMN were able to respond functionally to IL-2 by enhanced growth-inhibitory activity against an opportunistic fungal pathogen, Candida albicans. IL-2 activation of antifungal activity was dose-dependent, with some functional activation detected at 1 U/ml of rIL-2 and maximal activation at 1000 U/ml. The action of IL-2 was rapid, with maximal PMN activation after 30-min incubation with IL-2. The IL-2 enhancement of antifungal activity could be blocked by a specific antibody against IL-2R beta, but not by anti-IL-2R alpha. Analysis of the mechanism of IL-2 activation of PMN indicated that oxidative metabolism, as measured by superoxide anion production, was not involved. Instead, PMN release of lactoferrin appeared to be responsible for the heightened activity against C. albicans in IL-2-treated PMN. Not only was lactoferrin detected in the supernatants of IL-2-treated PMN, but also the antifungal activity of PMN activated by IL-2 could be blocked in the presence of antilactoferrin. These results, taken together, indicate that normal PMN are capable of functionally responding to IL-2 via expression of the IL-2R beta chain.

Inactivation of alpha-1-proteinase inhibitor by neutrophil metalloproteinases. Crucial role of the myeloperoxidase system and effects of the anti-inflammatory drug nimesulide.

Supernatants, obtained from normal neutrophil polymorphonuclear leukocytes (PMN), challenged with opsonized zymosan (OPZ), were found to inactivate the PMN elastase inhibitor, alpha 1-proteinase inhibitor (A1PI). As the supernatants were treated with methionine to quench residual oxidants, primarily chloramines, the observed inactivation of A1PI appears to be due to enzymes. The activity of the supernatants was in fact inhibited by metal-chelators and by the tissue inhibitor of metalloproteinases (TIMP), which is consistent with the intervention of metalloproteinases. Supernatants from normal PMN triggered by OPZ in the presence of inhibitors of the myeloperoxidase (MPO) system as well as supernatants from MPO-deficient PMN were inactive but displayed the capacity of inactivating A1PI after treatment with the metalloproteinases activator 4-aminophenylmercuric acetate. These data suggest that the A1PI inactivation is due to metalloenzymes released by PMN as latent molecules, in turn activated by the MPO system. The MPO-dependent autoactivation of latent metalloenzymes by PMN, with consequent A1PI inactivation, was inhibited by the nonsteroidal anti-inflammatory drug nimesulide (NMS). As PMN-derived HOCl is well known to inactivate A1PI directly, through a process previously shown to be inhibitable by NMS, the present results suggest: (1) both the oxidative and proteolytic inactivation of A1PI depend on the HOCl-generating MPO system; (2) the tissue-destructive activity of PMN elastase could be controlled by interfering pharmacologically with the PMN-MPO system, directly and indirectly responsible for the breakdown of the tissue antielastase screen.

Expression of the 50-kDa integrin-associated protein on myeloid cells and erythrocytes.

Integrin-associated protein (IAP) is a 50-kDa intrinsic membrane protein that is involved in signal transduction during neutrophil activation by a variety of Arg-Gly-Asp-containing ligands. However, IAP does not itself directly bind these ligands, which are instead recognized by the leukocyte response integrin (LRI). In fact, IAP is more widely expressed than the LRI and is even on erythrocytes, which express no known integrins. This suggests that IAP may have additional functions besides signal transduction in association with the LRI. In this work we have quantitated IAP expression on several myeloid cells and cell lines, as well as erythrocytes, using a mAb which recognizes this protein. These data show that there are about 10,000 IgG-binding sites on each erythrocyte and 20 times that number on the myeloid cell lines U937 and HL60. Normal PMN and monocytes express about 25,000 IgG-binding sites. There are twice as many Fab'-binding sites on each cell, suggesting that each IgG binds via both Ag-combining sites. Binding data using several mAb to IAP suggest that IAP undergoes a temperature-dependent conformational change. Unlike some integrin receptors, IAP is not stored in a regulated secretory compartment in polymorphonuclear leukocytes (PMN). In addition, there is no evidence for internalization or shedding of IAP upon PMN activation. These data show that IAP is expressed at significant levels on myeloid cells and erythrocytes and that its expression is unaffected by the state of PMN activation.

Effect of zymosan-activated plasma on the deformability of rabbit polymorphonuclear leukocytes.

Intravascular infusion of inflammatory mediators causes a sudden neutropenia due to the sequestration of polymorphonuclear leukocytes (PMN) within the microvasculature of the lung and other organs. This sequestration could be due to a decrease in the ability of PMN to deform and pass through the narrow capillary bed. The purpose of this study was to determine if the complement fragments present in zymosan-activated plasma (ZAP) caused a rapid stiffening of PMN. The PMN deformability was determined by measuring the pressure required to pass PMN through a polycarbonate filter containing 5-micron pores at a constant flow rate as well as the extraction of PMN compared with red blood cells and 125I-labeled albumin by the filter. The role of the cytoskeleton in PMN deformation was examined in studies where F-actin formation was inhibited using cytochalasin B or microtubule assembly was inhibited using colchicine. The results showed that treatment with ZAP induced a rapid decrease in PMN deformability. Inhibiting the formation of F-actin made the unstimulated PMN more deformable and reduced the stiffening induced by ZAP. In contrast, inhibition of microtubule reassembly did not alter either normal deformability or the ZAP-induced decrease in deformability. In vivo, colchicine increased normal PMN margination but did not inhibit the rapid sequestration of PMN induced by infusion of ZAP. These studies indicate that ZAP induces a rapid decrease in PMN deformability that is mediated through the cytoskeleton. They suggest that this decrease is due to the polymerization of F-actin.

Abnormal cytoplasmic pH regulation during activation in uremic neutrophils.

Neutrophils isolated from ESRF patients demonstrated abnormal cytoplasmic pH changes after FMLP stimulation; the initial cytoplasmic acidification was absent (P less than 0.001 compared to controls) and the degree of alkalinization enhanced (P less than 0.05 compared to controls). This effect was not due to the absence of any of the factors associated with acidification in normal PMN since superoxide production was enhanced (P less than 0.05 compared to controls) and intracellular calcium release was normal. Our observations are not explicable by alterations in the function of the Na:H antiport since the kinetics of antiport activation by cytoplasmic pH were not different in uremic and control cells. Other factors must therefore be important in generating the abnormal pH response to chemotactic factors in uremic PMN. Cells from CAPD patients had some degree of initial acidification (P less than 0.001 compared to controls and P less than 0.05 compared to ESRF) and enhanced alkalinization (P less than 0.05 compared to controls). Preincubation of normal PMN in four-hour dwell PDE reproduced the responses of uremic PMN with absent acidification, enhanced alkalinization and enhanced superoxide generation after FMLP stimulation (P less than 0.05 compared to controls). Changes in the control of cytoplasmic pH in stimulated PMN may influence PMN function, and our observations may be relevant to the susceptibility of uremic patients to infection.

Viricidal effect of polymorphonuclear leukocytes on human immunodeficiency virus-1. Role of the myeloperoxidase system.

Myeloperoxidase (MPO), H2O2, and chloride form an antimicrobial system in neutrophilic polymorphonuclear leukocytes (PMN) effective against a variety of microorganisms. Normal human PMN, when stimulated with phorbol myristate acetate or opsonized zymosan, are viricidal to HIV-1. The viricidal effect was lost when chloride was replaced by sulfate and was inhibited by the peroxidase inhibitor azide and by catalase, but not by heated catalase or superoxide dismutase, implicating H2O2. Stimulated PMN from patients with chronic granulomatous disease (CGD) were not viricidal to HIV unless H2O2 or glucose oxidase (which generates H2O2) was added, and the viricidal activity of H2O2-supplemented CGD PMN was inhibited by azide, implicating endogenous MPO. Stimulated PMN from patients with hereditary MPO deficiency had decreased viricidal activity unless MPO was added, and the viricidal activity of MPO-supplemented, MPO-deficient PMN was inhibited by catalase, implicating endogenous H2O2. The data suggest that when PMN are stimulated, MPO released by degranulation reacts with H2O2 formed by the respiratory burst to oxidize chloride to a product (presumably hypochlorous acid) that is toxic to HIV-1. Our findings raise the possibility that this viricidal effect of stimulated PMN may influence the host defense against HIV-1.

The effect of histamine on polymorphonuclear leukocyte-induced pneumocyte injury in vitro.

Background: Although polymorphonuclear leukocytes (PMN) are important in protecting the airways and alveolar surfaces, there is evidence that they can also injure the lung while exercising their defensive role. However it has been unclear whether PMN-induced pneumocyte injury is mediated by their direct cytotoxic effect on target cells or by PMN-derived cytotoxic mediators. On the other hand histamine was known not only to act as an important chemical mediator participated in the pathogenesis of some atotic and allegic disorders, but also to have an inhibitory effect on normal PMN functions. Method: To study the mechanism by which PMN induce pneumocyte injury, we cocultured PMN from four healthy nonsmokers or their PMN-derived supernatants (PMN-SPN) with monolayers of -labeled human A549 pneumocytes and compared PMN-and PMN-SPN-mediated pneumocyte injuries measured by release assay. We also compared the effects of histamine on each pneumocyte injury. Results: 1) PMN-SPN showed more injurious effect on A549 pneumocytes than that of PMN itself regardless histamine pretreatment of PMN. 2) Pneumocyte injury by PMN with histamine pretreatment was increased or decreased compared with that by PMN without histamine pretreatment, according to histamine concentrations, and PMN stimulating agents and their concentrations. 3) Pneumocyte injury by PMN-SPN with histamine pretreatment tended to be decreased compared with that by PMN-SPN without histamine pretreatment. Conclusion: Our results suggest that PMN-SPN may play more important role in mediating pneumocyte injury than PMN itself and that histamine may partially play a protective role on PMN-induced pneumocyte injury. Alternatively we conclude that the effects of histamine on PMN-induced pneumocyte injury may be affected by microenvironment in vivo.

Potency of antileukoprotease and alpha 1-antitrypsin to inhibit degradation of fibrinogen by adherent polymorphonuclear leukocytes from normal subjects and patients with chronic granulomatous disease.

We have studied the relative efficacy of antileukoprotease (ALP) and alpha 1-antitrypsin (alpha 1AT) to inhibit the degradation of substrate by polymorphonuclear leukocytes (PMN) attached onto a fibrinogen matrix. PMN elastase activity was assayed by radioimmunoassay of a specific 21-residue cleavage product from the amino terminus of the A alpha chain, A alpha (1-21), of fibrinogen. The adherence of PMN (1.0 x 10(6)) to a fibrinogen matrix was facilitated by incubation with recombinant tumor necrosis factor-alpha (1 nM). Subsequently, the cells were exposed to inhibitors before stimulation with cytochalasin B and formylmethionyl-leucylphenylalanine. Under these conditions, ALP inhibited A alpha (1-21) formation with an IC50 of 85 +/- 30 nM and alpha 1AT gave an IC50 of 220 +/- 98 nM (mean +/- SD). The effect of oxidant production on A alpha (1-21) formation was evaluated by comparing the effect of PMN from normal subjects with PMN from subjects with X-linked NADPH oxidase deficiency. Stimulation of PMN from the latter subjects in a similar fashion as described above resulted in the formation of 40 +/- 4 pmol/ml A alpha (1-21), or approximately twice the amount seen with cells from normal subjects. Preincubation with ALP or alpha 1AT in a concentration range between 10 to 900 nM resulted in an IC50 of 50 +/- 13 nM for ALP compared with 150 +/- 21 nM for alpha 1AT. Both inhibitors are more effective to prevent fibrinogen degradation caused by chronic granulomatous disease (CGD) PMN than by normal PMN despite the fact that CGD PMN generated more A alpha (1-21) than did normal PMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxin suppresses matrix protein-induced upregulation of PMN candicidal activity: An effect reversed by low-dose TNF-α

We investigated the relationship of polymorphonuclear leukocyte (PMN) candicidal activity, matrix proteins, and lipopolysaccharide (LPS) to determine how LPS modulates the normal enhancing effect of matrix proteins on PMN candicidal activity. LPS reduced PMN candicidal activity when PMN were adhered in the presence of either fibronectin or laminin. In the presence of fibronectin or laminin, LPS reduced CD11b/CD18 expression (the fibronectin receptor) as assessed using sheep erythrocytes coated with C3bi. Experiments with 125I-fibronectin and 125I-RGDS (ArgGlyAspSer) demonstrated that LPS reduced both the binding of fibronectin and the bioavailability of the binding epitope on the PMN surface. Stimulating the PMN oxidative burst with PMA but not FMLP also reduced fibronectin and RGDS binding. Incubation of LPS-treated PMN with staurosporine blocked the decrease in fibronectin and RGDS binding. Exposure of PMN to LPS plus low-dose TNF-α restored both fibronectin and RGDS binding with a concomitant increase in CD11b/CD18 surface expression. Low-dose TNF-α restored PMN candicidal activity in the presence of LPS and was most effective if PMN were preadhered to fibronectin. These results demonstrate that: (1) matrix proteins enhance normal PMN candicidal activity, (2) LPS reduces PMN candicidal activity in the presence of matrix proteins, (3) stimulation of the PMN oxidative burst in particular via protein kinase c activation reduces the bioavailability of the fibronectin receptor, and (4) low-dose TNF-α may restore PMN candicidal activity in part by upregulating the surface receptor for fibronectin binding.

Absolute requirement of CD11/CD18 adhesion molecules, FcRII and the phosphatidylinositol-linked FcRIII for monoclonal antibody-mediated neutrophil antihuman tumor cytotoxicity

We have previously shown that 3F8, a murine IgG3, monoclonal antibody (MoAb) specific for the ganglioside GD2, mediates tumor cell kill in vitro and in vivo. We now describe receptor requirements of polymorphonuclear leukocytes (PMN) in 3F8-mediated cytotoxicity (ADCC) of human GD2 (+) melanoma and neuroblastoma cell lines. PMN from a child with leukocyte adhesion deficiency (LAD) were devoid of CD11/CD18 adhesion molecules and mounted no detectable ADCC. MoAb to CD11b, CD11c, and CD18 each efficiently blocked ADCC by normal PMN. In contrast, a panel of different MoAbs to CD11a had no significant inhibitory effect on ADCC, a finding consistent with the low-to-absent expression of the CD11a ligand, intercellular adhesion molecule-1, on the target cells. Granulocyte-macrophage colony-stimulating factor (GM- CSF) significantly increased the expression of CD11b, CD11c, and CD18 on normal PMN, decreased the expression of Fc receptors (FcR), and enhanced ADCC by normal but not by LAD PMN. MoAbs to FcRII and FcRIII each efficiently blocked ADCC; anti-FcRI MoAb had no effect. Flow cytometry using anti-FcRII MoAb versus anti-FcRIII MoAb did not show cross competition, suggesting that inhibition of ADCC was not a steric effect resulting from FcRII proximity to FcRIII. PMN deficient in FcRIII (obtained from patients with paroxysmal nocturnal hemoglobinuria) and PMN depleted of FcRIII by treatment with elastase or phosphatidylinositol (PI)-specific phospholipase C produced low ADCC, supporting a role for the PI-liked FcRIII. Thus, optimal ADCC using human PMN, human solid tumor cells, and a clinically active MoAb (conditions that contrast with the heterologous antibodies and nonhuman or nonneoplastic targets used in most models of PMN ADCC) required CD11b, CD11c, FcRII, and the PI-linked FcRIII. Furthermore, in this clinically relevant system, GM-CSF enhancement of antitumor PMN ADCC correlated with increased expression of CD11/CD18 molecules.

Absolute Requirement of CD11/CD18 Adhesion Molecules, FcRII, and the Phosphatidylinositol-Linked FcRIII for Monoclonal Antibody-Mediated Neutrophil Antihuman Tumor Cytotoxicity

We have previously shown that 3F8, a murine IgG3, monoclonal antibody (MoAb) specific for the ganglioside GD2, mediates tumor cell kill in vitro and in vivo. We now describe receptor requirements of polymorphonuclear leukocytes (PMN) in 3F8-mediated cytotoxicity (ADCC) of human GD2 (+) melanoma and neuroblastoma cell lines. PMN from a child with leukocyte adhesion deficiency (LAD) were devoid of CD11/CD18 adhesion molecules and mounted no detectable ADCC. MoAb to CD11b, CD11c, and CD18 each efficiently blocked ADCC by normal PMN. In contrast, a panel of different MoAbs to CD11a had no significant inhibitory effect on ADCC, a finding consistent with the low-to-absent expression of the CD11a ligand, intercellular adhesion molecule-1, on the target cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased the expression of CD11b, CD11c, and CD18 on normal PMN, decreased the expression of Fc receptors (FcR), and enhanced ADCC by normal but not by LAD PMN. MoAbs to FcRII and FcRIII each efficiently blocked ADCC; anti-FcRI MoAb had no effect. Flow cytometry using anti-FcRII MoAb versus anti-FcRIII MoAb did not show cross competition, suggesting that inhibition of ADCC was not a steric effect resulting from FcRII proximity to FcRIII. PMN deficient in FcRIII (obtained from patients with paroxysmal nocturnal hemoglobinuria) and PMN depleted of FcRIII by treatment with elastase or phosphatidylinositol (PI)-specific phospholipase C produced low ADCC, supporting a role for the PI-liked FcRIII. Thus, optimal ADCC using human PMN, human solid tumor cells, and a clinically active MoAb (conditions that contrast with the heterologous antibodies and nonhuman or nonneoplastic targets used in most models of PMN ADCC) required CD11b, CD11c, FcRII, and the PI-linked FcRIII. Furthermore, in this clinically relevant system, GM-CSF enhancement of antitumor PMN ADCC correlated with increased expression of CD11/CD18 molecules.

Characterization of a direct effect of phorbol myristate acetate on human neutrophil cell membrane using 31D8 monoclonal antibody.

We found that 4-beta-phorbol 12-myristate 13-acetate (PMA) caused decreased expression of the polymorphonuclear neutrophil (PMN) surface antigen 31D8. In contrast to the rapid initiation of the oxidative burst caused by PMA, the effect was slow to start but increased during incubation periods up to 50 min. To study this apparent protein kinase C-independent late effect of PMA, we measured 31D8 expression in PMNs after incubation with various concentrations of PMA. The maximum PMA-induced inhibition was 76 +/- 2%, with an ID50 of 3.9 +/- 0.4 ng/ml. Oxidants and prooxidants (hydrogen peroxide, hypochlorite, taurine-chloramine, and ferrous iron, with or without H2O2) had no direct effect on 31D8 antigen expression. The following substances were not protective against the inhibitory affect of PMA: (1) antioxidants (superoxide dismutase, catalase, azide, dimethyl sulfoxide, Desferal, and ascorbate, with the exception of alpha-tocopherol), (2) inhibitors of protein kinase C (H7 and W7), (3) inhibitors of 5-lipoxygenase (A-63162, MK886, and high-dose indomethacin) and (4) inhibitors of cyclooxygenase (low-dose indomethacin). Myeloperoxidase-deficient PMNs had normal 31D8 antigen expression and a decrease of 31D8 antigen expression by PMA, as did normal PMNs. The inactive analog of PMA, 4-alpha-phorbol didecanoate, had no effect on 31D8 antigen expression. alpha-Tocopherol (50 micrograms/ml) and betamethasone (150 micrograms/ml) protected against the PMA effect by 30.5 +/- 7.3 (P less than .0005) and 52 +/- 15 (P less than 0.004) channels, respectively. These results indicate that PMA has a protein kinase C-independent late effect on human neutrophils, which can be prevented by pretreatment with alpha-tocopherol or the steroid betamethasone. These compounds probably exert their protective effect by membrane stabilization.

The glycosylphosphatidylinositol-linked Fc gamma receptor III represents the dominant receptor structure for immune complex activation of neutrophils.

Polymorphonuclear neutrophils (PMN) express constitutively two low-affinity Fc gamma receptors, Fc gamma RII and Fc gamma RIII. Fc gamma RII is a transmembrane molecule, and Fc gamma RIII is linked via a glycosylphosphatidylinositol (GPI) anchor to the membrane. The role of each of these receptors in activation of PMN is still unclear. We used specific cross-linking of Fc gamma RII via Fab fragments of IV.3 (anti-Fc gamma RII, CDw32) and of Fc gamma RIII using F(ab')2 fragments of 3G8 (anti-Fc gamma RIII, CD16) to activate PMN. Stimulation of Fc gamma RIII was significantly more effective in inducing a respiratory burst than cross-linking of Fc gamma RII. A synergistic effect was observed after simultaneous activation of Fc gamma RIII. We could demonstrate that both Fc gamma R mobilize calcium as intracellular signal in spite of their different membrane linkage. The kinetic of calcium mobilization after Fc gamma R stimulation is delayed in comparison to formyl-methionyl-leucyl-phenylalanine activation. In addition Fc gamma R-induced increase of cytoplasmic calcium is pertussis toxin insensitive. When monoclonal IgG1 kappa complexes were used for stimulation calcium mobilization and hydrogen peroxide (H2O2) production could also be demonstrated. Inhibition studies of this activation using monoclonal antibodies suggested that this immune complex activation was predominantly mediated via Fc gamma RIII. Only in Fc gamma RIII-deficient PMN from paroxysmal nocturnal hemoglobinuria patients could a decreased H2O2 production be demonstrated to be Fc gamma RII dependent. In normal PMN the GPI-anchored Fc gamma RIII structure is the predominant receptor.

Increased spontaneous release of cytidine deaminase by polymorphonuclear neutrophils of patients with active systemic lupus erythematosus.

Cytidine deaminase activity (CD) in the neutrophil culture supernatants (PMN SUP) of 27 patients with systemic lupus erythematosus (SLE) was measured using a spectrophotometric method. Compared with the controls (5.449 +/- 1.358 U/5 x 10(6) PMN), the CD activity in the spontaneous culture supernatants of PMN was significantly increased in active (10.003 +/- 2.637 U/5 x 10(6) PMN) but not in inactive (5.358 +/- 1.624 U/5 x 10(6) PMN) SLE. However, after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 x 10(-7) M), the ratio of enzyme activity between stimulated and spontaneous PMN supernatants was decreased in active SLE (0.794 +/- 0.178) compared with normal controls (1.300 +/- 0.225). In contrast, the enzyme activity in the cytoplasm of either stimulated or non-stimulated PMN was not different among these three groups. These results suggest that CD of PMN is releasable and can be enhanced by chemotactic factor stimulation in normal PMN. The increased spontaneous release of CD by active SLE PMN is one of the indicators for the disease activity in these patients.

Evaluation of prostaglandin E 1 for prevention of respiratory failure in high risk trauma patients: A prospective clinical trial and correlation with plasma suppressive factors for neutrophil activation

A group of 48 critically injured patients were entered into a prospective, double-blind, placebocontrolled trial to evaluate the efficacy of early infusion of PGE 1 for reducing the incidence of severe respiratory failure and mortality. Secondary assessments examined the effects of the PGE 1 infusion on plasma mediated suppression of PMN superoxide production and loss of PMN granule enzyme content. The incidence of severe respiratory failure was lower in the PGE 1 group — 13% versus 32%, but this did not reach significance. The overall morality was equivalent between the two groups — 26% (PGE 1) versus 28% (placebo). The suppressive activity of the patient plasma was assayed by measurement of normal PMN superoxide production relative to normal control plasma (ratio P:C). The baseline ratio P:C was 62 ± 5% in the PGE 1 group versus 60 ± 5% in the placebo group. The day 1 plasma samples showed significant reversal of plasma suppressive activity in the PGE 1 group — ratio P:C 88 ± 5% versus 67 ± 5% in the placebo group (P < 0.02). In patients who received the full 7 days of infusion, the plasma suppressive activity remained significantly diminished in the PGE 1 group — ratio P:C 77 ± 4% versus 61 ± 5% (P < 0.04). The baseline lysozyme content of patient PMN's relative to that of normal control PMNs (ratio P:C) was 119 ± 14% in the PGE 1 group. A significant loss of lysozyme content was observed in the PGE 1 group on day 1 of the infusion — ratio P:C 79 ± 8% (P < 0.03), and was associated with a reduction in the plasma suppressive activity. Although no significant clinical benefit could be attributed to the PGE 1 infusion, these studies indicate that infusion of PGE 1 was associated with attenuated production of post traumatic plasma suppressive factors of PMN activation.

Increased expression of the interleukin 1 receptor on blood neutrophils of humans with the sepsis syndrome.

Because of the potential importance of interleukin 1 (IL-1) in modulating inflammation and the observations that human blood neutrophils (PMN) express IL-1 receptors (IL-1R) and synthesize IL-1 alpha and IL-1 beta, we studied the IL-1R on blood PMN from a group of patients with the sepsis syndrome. We report a marked enhancement in the sites per cell of IL-1R expressed on sepsis-PMN of 25 consecutively studied patients compared to 20 controls (patient mean = 9,329 +/- 2,212 SE; control mean = 716 +/- 42 SE, respectively). There was no demonstrable difference in the Kd of IL-1R on sepsis-PMN (approximately 1 nM) as determined by saturation curves of 125I-IL-1 alpha binding and the IL-1R on sepsis-PMN had an apparent Mr approximately 68,000, a value like that of normal PMN. Cytofluorographic analysis indicated that the sepsis-PMN phenotype is a single homogeneous population with respect to IL-1R expression. In contrast, expression of the membrane complement receptor CR3 is not increased on sepsis-PMN. Similar increases in expression of IL-1R were not observed in various other inflammatory processes, including acute disseminated inflammation and organ failure not caused by infection, acute infection without organ failure, and immunopathologies such as active systemic lupus erythematosus and rheumatoid arthritis. Enhanced expression of IL-1R was not related simply to the state of myeloid stimulation. Increased expression of IL-1R on normal PMN was induced in vitro by incubating cells with recombinant human granulocyte-macrophage/colony-stimulating factor for 18 h and this response was inhibited by cycloheximide, suggesting the possibility that de novo synthesis of IL-1R might occur in PMN during the sepsis syndrome.

Neutrophils from chronic granulomatous disease fail to increase endothelial permeability.

Studies of neutrophil-dependent endothelial injury were made using normal neutrophils (nl-PMN, i.e., normal polymorphonuclear neutrophils) and PMN obtained from a patient with X-linked chronic granulomatous disease (CGD-PMN). Layering of nl-PMN on bovine pulmonary microvessel endothelial monolayers (ratio 10:1) followed by challenge with phorbol 12-myristate 13-acetate (PMA; 5 x 10(-9) M) resulted in a 190% increase in 125I-labeled albumin permeability across the monolayers. Pretreatment of endothelial monolayers with tumor necrosis factor-alpha (TNF-alpha; 200 or 1,000 U/ml) further enhanced the increase in permeability after stimulation of nl-PMN with PMA. In contrast, challenge of CGD-PMN layered on endothelial cells with PMA did not increase permeability, and the effect was not enhanced by pretreating the endothelial cells with TNF-alpha. Nl-PMN, but not CGD-PMN, generated superoxide anion on stimulation with PMA (42.5 +/- 0.7 vs. 6.5 +/- 0.6 nM.10(6) PMN-1.h-1). PMA also induced degranulation of nl-PMN as measured by release of elastase (2.32 +/- 0.12 micrograms/10(6) PMN) and myeloperoxidase (173.8 +/- 1.9 U/10(6) PMN) into the medium, whereas release by CGD-PMN was markedly reduced (elastase release of 0.88 +/- 0.04 microgram/10(6) PMN and myeloperoxidase release of 82.6 +/- 19.4 U/10(6) PMN). The adherence response of nl-PMN and CGD-PMN to the endothelial cells after PMA stimulation was similar (79.5 +/- 11.8% nl-PMN and 64.0 +/- 1.9% CGD-PMN). Therefore, the PMN respiratory burst, with the resultant generation of oxidants and PMN-derived proteases, is required to increase endothelial permeability even in the face of increased endothelial adhesivity.

The effects of IL-1, IL-2, and tumor necrosis factor on polymorphonuclear leukocyte Fc gamma receptor-mediated phagocytosis. IL-2 down-regulates the effect of tumor necrosis factor.

It has been reported that the Fc gamma R-mediated phagocytic activity of polymorphonuclear leukocytes (PMN) from patients with acute bacterial infections is markedly enhanced when compared with healthy controls. Inasmuch as several potent cytokines are known to be involved in inflammatory and infectious processes, we studied the effects of three such cytokines (IL-1 beta, IL-2, and TNF-alpha) on normal PMN Fc gamma R-mediated phagocytosis. IL-1 beta and TNF alpha both caused a significant increase in the ingestion of EIgG by adherent PMN. In combination, IL-1 beta and TNF-alpha had an additive effect, even when each was used at its optimal concentration. In contrast to the enhancing effects mediated by IL-1 beta and TNF-alpha, IL-2 alone had no significant effect on PMN phagocytosis. Notably, however, IL-2 at a concentration of 10(4) U/ml partially inhibited TNF-alpha-mediated enhancement of phagocytosis by decreasing TNF binding to the PMN cell surface. This inhibitory effect of IL-2 on TNF was reversed by anti-IL-2 antibody and mAb directed against the low affinity IL-2R (anti-Tac), whereas mAb directed against the intermediate affinity receptor (mik-beta 1) had no such effect. These findings may have important physiologic implications, because patients receiving IL-2 therapy have been shown to have increased susceptibility to infection.