The influence of net surface charge on the interaction of uropathogenic Escherichia coli with human neutrophils

Escherichia coli strains, grown to suppress fimbrial expression, synthesised enhanced quantities of polysaccharide capsule, which significantly lessened their binding to heparin sepharose columns. In the presence of poly-L-lysine, these strains were strongly retained on the columns confirming their highly anionic nature. Uropathogenic strains of E. coli expressing type 1 fimbrial adhesins activated the respiratory burst, the degranulation response and the release of leukotrienes from human neutrophils (PMN) to a significantly greater extent than the same strains grown in a medium to suppress this fimbrial expression. The addition of the poly-cation poly-L-lysine, however, selectively increased neutrophil activation in response to these non-fimbriate strains. This dose-dependent effect was reversed by the addition of heparin suggesting a mechanism dependent on surface charge. The results of this study suggest that non-specific mechanisms involving the neutralisation of surface charge, in addition to specific receptor and adhesin mediated events could affect neutrophil activation at sites of infection.     

Enhanced killing of Pseudomonas aeruginosa by human polymorphonuclear leukocytes in presence of subinhibitory concentrations of carbenicillin and ticarcillin

The effect of carbenicillin and ticarcillin on the killing of Pseudomonas aeruginosa was studied with an in vitro system using peripheral blood polymorphonuclear (PMN) leukocytes collected from human donors. No corticosteroid was given to the donor prior to leukocytes collection by a continuous flow cell separator. The assay was carried out with or without serum. P. aeruginosa yield after a 4 hour-incubation was estimated by colony counting. In Hanks' balanced salt solution, P. aeruginosa strains 74 and 78 were resistant to human PMN leukocytes. The presence of subinhibitory concentrations of carbenicillin or ticarcillin (1/10th the minimal inhibitory concentration (MIC) for P. aeruginosa 74, 1/4th the MIC for P. aeruginosa 78) enhanced the bactericidal activity of human leukocytes. Difference between the numbers of bacteria recovered with PMN cells and without cells increased with concentration of carbenicillin or ticarcillin. The synergistic effect was not observed when serum (heated fetal calf serum or heated pooled human serum) was used. The mode of action of carbenicillin and ticarcillin on bactericidal activity of phagocytic cells was not elucidated, but we suggest the effect is due not to action on the phagocytic cells themselves but on the microorganisms.     

Polymorphonuclear granulocytes induce antibody-dependent apoptosis in human breast cancer cells

Recent studies in HER-2/neu-targeted immunotherapy demonstrated that polymorphonuclear neutrophils (PMN) mediated Ab-dependent cellular cytotoxicity against HER-2/neu-positive breast cancer cell lines. However, the mechanism of cell death remained unclear. We used several assays to analyze the induction of apoptosis in the breast cancer cell line SK-BR-3 via PMN-dependent Ab-dependent cellular cytotoxicity. In the presence of the HER-2/neu Ab 520C9 and PMN from healthy donors, apoptosis occurred as detected by annexin V binding and disappearance of euploid SK-BR-3 nuclei, which can be differentiated from PMN nuclei by their increased DNA contents. Apoptosis induction was observed with E:T cell ratios as low as 10:1. Laser scanning fluorescence microscopy of TUNEL tumor cells or staining for cleaved cytokeratin-18 further confirmed apoptosis of the SK-BR-3 breast cancer cells. Killing via 520C9 was dependent on the interaction with FcR on PMN, because 1) F(ab')(2) fragments of 520C9 mediated no cytotoxicity, 2) target cell death was influenced by a biallelic polymorphism of FcgammaRIIa on the effector cells, and 3) a bispecific Ab against HER-2/neu and the IgA receptor (FcalphaRI) expressed on effector cells significantly induced apoptosis. Thus, PMN induce Ab-dependent apoptosis against human breast cancer cells targeted with HER-2/neu-directed mAbs or FcR directed bispecific Abs.     

Human IgA1 initiates complement-mediated killing of Neisseria meningitidis

We studied the effect of human IgA1, the predominant IgA subclass in serum, on C-mediated killing of Neisseria meningitidis. We purified monomeric IgA1 from normal human serum and tetravalent meningococcal polysaccharide vaccinate serum by using the following successive chromatographic steps: jacalin lectin affinity, Superose 12 FPLC gel filtration, Mono Q FPLC anion exchange, and anti-IgG affinity. SDS-PAGE, ELISA, and Western immunoblot analyses of the IgA1 detected no trace of contaminating IgG or IgM. IgA1 initiated partial or complete lysis (62 to 100%) of nine group C strains by using either normal, hypogammaglobulinemic, factor B-depleted, or properdin-deficient human serum as a C source, but IgA1 was unable to effect killing in serum chelated with 10 mM MgCl2 and 10 mM EGTA. Lytic activity was dependent on the group C strain and the source of the IgA1; neither IgA1 preparation was bactericidal for all nine strains. Removal of the Fc portion of IgA1 with pepsin completely abolished bactericidal activity. We purified and radiolabeled C component C3, and found that IgA1 did not increase C3 deposition. With the use of a group C polysaccharide ELISA, we found that the vaccinate IgA1 had a high titer of group C polysaccharide antibody, whereas the IgA1 purified from normal human serum had no detectable group C polysaccharide specificity. Absorption of the vaccinate IgA1 with alum-bound group C polysaccharide did not affect the killing of a sensitive strain, but it did potentiate the killing of a previously resistant strain. Western immunoblots of whole cell lysates, outer membrane complex, and purified lipooligosaccharide showed that the bactericidal IgA1 was specific for several outer membrane proteins. Four of the proteins recognized by both IgA1 preparations had apparent Mr of 29, 42, 66, and 74 kDa. We conclude that IgA1, when bound to specific outer membrane proteins, can initiate lysis of group C meningococci via the classical C pathway, and that initiation of lysis is an Fc-dependent event which occurs without an increase in C3 deposition.     

Cytochemical investigation of neutral proteases in polymorphonuclear (PMN) neutrophils in acute inflammatory diseases

Summary Neutral proteases can be released from PMN neutrophils in blood smears from healthy subjects by incubation with NaCl-borate buffer. The activity of the PMN proteases can be revealed by the degradation of erythrocytes and plasma within ring-shaped areas centered around each neutrophil (halo effect). During the acute stage of various inflammatory diseases (pneumonia, meningitis, cholecystitis, etc.) the activity of neutral PMN proteases is substantially reduced, as reflected by reduced halo formation. After recovery, halo formation returns to normal. Temporary lowering of neutral PMN proteases is thus one of a series of functional defects of PMN neutrophils which are detectable in the course of acute infectious diseases. These include reduced phagocytosis, altered chemotaxis and reduced bactericidal function. The cytochemical test for neutrophilic granulocyte function used in the present investigation is especially practical by comparison with the other techniques: it saves time and is simple to perform.Dedicated to Prof. W. Graumann on the occasion of his 65th birthday     

Immunofluorescence analysis of IgA binding by human mononuclear cells in blood and lymphoid tissue

The nature of IgA-binding cells and their tissue distribution was examined by an indirect immunofluorescence assay with the use of IgA1 and IgA2 paraproteins and fluorochrome- or biotin-labeled F(ab')2 fragments of idiotype-specific antibodies. The frequency of IgA-binding mononuclear cells was approximately 13% in blood and spleen samples but less than 1% in tonsil samples. IgA binding could be visualized by flow immunocytometry on monocyte/macrophages, but not on T and B cells. IgA polymers were bound better than IgA dimers and monomers. Nonhomologous IgA myelomas of both IgA1 and IgA2 subclasses inhibited the IgA-binding to monocytes, whereas aggregated normal serum IgG, IgM paraproteins, and an IgG myeloma did not. IgA binding was relatively insensitive to changes in temperature or cation concentration. IgA-binding monocytes were found in IgA-deficient patients at the same frequency as in normal individuals. The results indicate that monocytes constitutively express class-specific binding sites for both IgA1 and IgA2 molecules.     

Stimulation of human neutrophil Fc receptor-mediated phagocytosis by a low molecular weight cytokine

Human neutrophil Fc receptor-mediated phagocytosis can be markedly enhanced by a low m.w. (less than 10,000) heat-labile cytokine(s) derived from specifically stimulated human mononuclear cells and from a human T cell line, MO(t). PMN incubated with supernatant from control mononuclear leukocyte (MNL) culture bound EIgG (percentage of rosettes = 73.7% +/- 7.1) but did not ingest the attached targets (phagocytic index, PI = 40.7 +/- 9.5) as efficiently as PMN incubated with supernatant from adherent MNL, which had ingested EIgG and were then cocultured with nonadherent MNL (PI = 264.3 +/- 46.3). Cytokine-containing supernatants were fractionated on YM-10 Centricon microconcentrators, and the effluent (YM-10E) was found to contain the phagocytosis-enhancing activity. Optimal Fc receptor-mediated ingestion by YM-10E-stimulated PMN required a critical level of target-bound IgG; stimulation was dose dependent and detectable after 5 min at 37 degrees C with a maximal response by 15 min. Monoclonal antibody 3G8 (anti-PMN Fc receptor) inhibited in a dose-dependent fashion both Fc receptor-mediated rosette formation and ingestion by nonstimulated and YM-10E-stimulated PMN. Solid-phase 3G8 Fab had the same effect. A previously undescribed monoclonal antibody, 1C2, exhibited a different pattern of inhibition. It had no effect on rosetting or ingestion of EIgG by nonstimulated PMN; however, it inhibited EIgG phagocytosis by YM-10E-stimulated PMN down to the level of nonstimulated ingestion without affecting rosette formation. Solid-phase 1C2 had the same effect. These data indicate that phagocytosis mediated by 3G8-positive Fc receptors may be enhanced by cytokine(s) stimulation in a manner requiring the molecule recognized by 1C2. Monoclonal antibodies to the alpha-chain of CR3 had only minimal effects on YM-10E-stimulated ingestion. Fluorescence flow cytometry of YM-10E-stimulated PMN, indirectly stained with 3G8 or 1C2, indicated that cytokine enhancement of EIgG ingestion occurred without an increase in either 3G8 or 1C2 binding sites. These data show that the low avidity Fc receptor, which binds immune complexes, may be functionally modulated at sites of inflammation where PMN and macrophages mediate clearance and destruction of immune complexes and opsonized particles.     

Normal human neutrophils are a source of a specific interleukin 1 inhibitor

In the course of our study on neutrophil production of an interleukin 1 (IL-1)-like factor, we found that the addition of polymorphonuclear neutrophils (PMN) to monocytes cultured in the presence of zymosan resulted in decreased IL 1 activity of the resultant supernatant, suggesting that PMN may contain an inhibitor of IL 1. The objective of this investigation was to study this IL 1 inhibitor which normal human PMN contain. The inhibitor is constitutively present in the PMN because 0 hr PMN lysates and unstimulated PMN supernatants also show inhibitory activity. The PMN inhibitor inhibits IL 1 (crude and partially purified) in a dose-response manner and does not affect basal [3H]thymidine incorporation in the presence or absence of PHA-P. The PMN inhibitor does not have any effect on interleukin 2 (IL 2)-induced proliferation of the IL 2-dependent CTLL cells. The inhibitor can be generated in the absence of serum and is not produced as a result of proteolytic activity from PMN enzymes. The inhibitor is heat-labile and is most stable at neutral pH. Gel filtration studies on Sephadex G-200 indicate that the inhibitor is heterogeneous in size. Two inhibitory peaks, at 45,000 to 70,000 m.w. and at greater than 160,000 m.w., were observed. When zymosan-stimulated PMN supernatant was chromatographed, there was separation of inhibitory factor from a 17,000 m.w. proliferating factor. Presence of this PMN inhibitor may be important in negative regulation of IL 1.     

Mechanism of priming of human neutrophils by a soluble lymphoblastoid cell factor

Treatment of human neutrophils (PMN) with a cytokine-like factor in the supernatants of human lymphoblastoid cells (Raji) increased the random mobility and enhanced the migration of treated cells in response to other chemoattractants nearly 21/2-fold, although the supernatant itself was not a chemoattractant. Supernatant treatment also increased the adherence of bacteria threefold and the bacterial killing fourfold compared with PMN treated with control media. In examining the metabolic basis for the enhanced bactericidal ability, we observed a significant increase in spontaneous hexose monophosphate shunt activity of Raji cell supernatant (RS)-treated neutrophils even in the absence of additional stimuli. RS-treated PMN also had significantly enhanced production of superoxide anion and chemiluminescence response upon subsequent stimulation with a variety of soluble and particulate stimuli. Unlike other agents that prime neutrophil activation, however, the factor(s) in RS did not cause degranulation. It also differed in its ability to progressively enhance PMN functions with a longer period of preincubation (up to 3 hr). These data suggest that the RS factor(s) primes neutrophils by a unique mechanism. The neutrophil-enhancing activities of RS, which are the opposite of those activities described for leukocyte inhibitory factor, eluted off a Sephacryl S-200 column at approximately 30,000 m.w. This factor expands the relationship between neutrophils and lymphocytes, and may be a useful agent to provide valuable insights into the mechanism of respiratory burst activation and regulation.     

Histamine modulates in vitro IgG production by pokeweed mitogen-stimulated human mononuclear cells

The specificity of the receptor for IgA (RFcα) on human peripheral blood monocytes and polymorphonuclear (PMN) cells was evaluated by the ability of various human IgA preparations to inhibit rosette formation between these cells and IgA-sensitized ox erythrocytes. RFc?α on PMNS and monocytes were blocked by both monomer and dimer IgA preparations indicating that multivalent expression of Fc regions does not play a major role in receptor binding and that neither secretory component nor J chain significantly influences the binding of RFcα to IgA. Immunoglobulins of both the IgA1 and IgA2 subclasses inhibited IgA rosette formation and were in fact quite similar in their efficiency of blocking of RFcα. An IgA paraprotein without a Cα₃ domain was an even better inhibitor of IgA rosette formation than the IgA1 or IgA2 immunoglobulins. This implicated the Cα₂ domain as the site on IgA which interacts with RFcα. Furthermore, that this Cα₃-deficient IgA, which exists as a half molecule, was very efficient at blocking RFcα also demonstrated that multivalent Fc expression is not important to binding of RFcα and moreover that the site on IgA which interacts with RFcα is not dependent on H-chain pairing. RFcα on both PMN cells and monocytes were susceptible to proteolysis by pronase at concentrations which did not affect the receptor for IgG on these cells. Within 18 hr after removal of RFcα these cells had resynthesized and displayed this receptor. Although it is unclear whether IgA alone can mediate the effector functions of PMNs and monocytes in mucosal areas, the present studies define more clearly the specificity and regenerative capacity of RFcα and provide a basis for understanding the role of receptors for IgA and the cells with which they are associated in immune defense especially on the mucosal surfaces.     

The specificity of the IgA receptor purified from human neutrophils.

A receptor for IgA was purified from human polymorphonuclear neutrophils (PMN) by affinity chromatography on human serum IgA-Sepharose. The receptor appeared on SDS/polyacrylamide gels as a diffuse band with an apparent molecular mass of 50-70 kDa, whether reduced or non-reduced. During purification, the protein showed remarkable stability to proteolytic digestion by endogenous PMN proteinases. Purified radioiodinated receptor re-bound to IgA-Sepharose, but not to IgG-Sepharose or BSA-Sepharose. The binding of the receptor to IgA-Sepharose was inhibited in a dose-dependent manner by human serum IgA1 or IgA2 or secretory IgA1 or IgA2, but not by IgG or IgM. Binding of receptor to IgA-Sepharose was also inhibited by the Fc fragment of IgA, but not by the Fab fragment. An IgA fragment produced by digestion with pepsin which lacks the CH3 domain also inhibited binding, but to a more limited extent than did the whole IgA molecule.     

Effector mechanisms of recombinant IgA antibodies against epidermal growth factor receptor

IgA is the most abundantly produced Ab isotype in humans, but its potential as immunotherapeutic reagent has hardly been explored. In this study, we describe anti-tumor mechanisms of mouse/human chimeric IgA Abs against the epidermal growth factor receptor (EGF-R). EGF-R Abs of IgG isotype are currently approved for the treatment of colon or head and neck cancers. As expected, the human IgG1, IgA(1), and IgA(2) variants of the 225 Ab demonstrated similar binding to EGF-R. Furthermore, IgA Abs were as effective as IgG in mediating direct effector mechanisms such as blockade of EGF binding, inhibition of EGF-R phosphorylation, and induction of growth inhibition. None of the three variants induced complement-mediated lysis. Human IgG1 effectively recruited MNC for ADCC, but activated PMN only weakly, whereas both IgA isoforms proved to be effective in triggering neutrophils. Interestingly, the IgA(2) isoform was significantly superior to its IgA(1) counterpart in recruiting PMN as effector cells. Because neutrophils constitute the most abundant effector cell population in human blood, this enhanced neutrophil recruitment lead to increased killing of EGF-R expressing tumor cells in whole blood assays. This killing was further enhanced when blood from G-CSF-primed donors was compared with healthy donor blood. Together, these data suggest EGF-R Abs of human IgA isotype to bear promise for therapeutic use in cancer.     

Effects of granulocyte-macrophage colony-stimulating factor and cyclic AMP interaction on human neutrophil apoptosis.

The current study was undertaken to evaluate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and cyclic AMP (cAMP) signaling interaction on human neutrophil apoptosis, either occurring spontaneously or induced by Fas antigen activation. Results show that GM-CSF, dibutyryl cAMP (a cAMP analog) and forskolin (an adenylate cyclase activator) are all able to suppress spontaneous neutrophil cell death. Of note however, when GM-CSF is used in combination with cAMP-elevating agents, an additive effect on neutrophil survival is observed with dibutyryl cAMP only, whereas supplementation of cell cultures with GM-CSF and forskolin results in a progressive reduction of antiapoptotic effects exerted by the single compounds. Moreover, although dibutyryl cAMP and forskolin do not affect Fas-triggered apoptotic events, they are still able to modulate the GM-CSF capacity to prolong neutrophil survival following anti-Fas IgM cell challenge, with effects similar to those respectively exerted on spontaneous neutrophil apoptosis. The data indicate that GM-CSF may negatively modulate the cAMP-mediated antiapoptotic pathway in human neutrophils, likely via the inhibition of adenylate cyclase activity. This would prevent an abnormal neutrophil survival as a result of cAMP signaling stimulation, which provides a novel insight into the role of GM-CSF as a physiological regulator of myeloid cell turnover.     

Respiratory syncytial virus infection of human lung endothelial cells enhances selectively intercellular adhesion molecule-1 expression

Respiratory syncytial virus (RSV) is worldwide the most frequent cause of bronchiolitis and pneumonia in infants requiring hospitalization. In the present study, we supply evidence that human lung microvascular endothelial cells, human pulmonary lung aorta endothelial cells, and HUVEC are target cells for productive RSV infection. All three RSV-infected endothelial cell types showed an enhanced cell surface expression of ICAM-1 (CD54), which increased in a time- and RSV-dose-dependent manner. By using noninfectious RSV particles we verified that replication of RSV is a prerequisite for the increase of ICAM-1 cell surface expression. The up-regulated ICAM-1 expression pattern correlated with an increased cellular ICAM-1 mRNA amount. In contrast to ICAM-1, a de novo expression of VCAM-1 (CD106) was only observed on RSV-infected HUVEC. Neither P-selectin (CD62P) nor E-selectin (CD62E) was up-regulated by RSV on human endothelial cells. Additional experiments performed with neutralizing Abs specific for IL-1alpha, IL-1beta, IL-6, and TNF-alpha, respectively, excluded an autocrine mechanism responsible for the observed ICAM-1 up-regulation. The virus-induced ICAM-1 up-regulation was dependent on protein kinase C and A, PI3K, and p38 MAPK activity. Adhesion experiments using polymorphonuclear neutrophil granulocytes (PMN) verified an increased ICAM-1-dependent adhesion rate of PMN cocultured with RSV-infected endothelial cells. Furthermore, the increased adhesiveness resulted in an enhanced transmigration rate of PMN. Our in vitro data suggest that human lung endothelial cells are target cells for RSV infection and that ICAM-1 up-regulated on RSV-infected endothelial cells might contribute to the enhanced accumulation of PMN into the bronchoalveolar space.     

The bacterial peptide N-formyl-Met-Leu-Phe inhibits killing of Staphylococcus epidermidis by human neutrophils in fibrin gels.

To study human neutrophil (polymorphonuclear leukocyte (PMN)) migration and killing of bacteria in an environment similar to that found in inflamed tissues in vivo, we have used fibrin gels. Fibrin gels (1500 microm thick) containing Staphylococcus epidermidis were formed in Boyden-type chemotaxis chambers. PMN migrated < 300 microm into these gels in 6 h and did not kill S. epidermidis when the gels contained heat-inactivated serum, C5-deficient serum, a streptococcal peptidase specific for a fragment of cleaved C5 (C5a), or anti-C5aR IgG. In contrast, in gels containing normal human serum, PMN migrated approximately 1000 microm into the gels in 4 h and into the full thickness of the gels in 6 h, and killed 90% of S. epidermidis in 6 h. fMLP reduced PMN migration into fibrin gels and allowed S. epidermidis to increase by approximately 300% in 4 h, whereas leukotriene B(4) stimulated PMN to migrate the full thickness of the gels and to kill 80% of S. epidermidis in 4 h. We conclude that both complement opsonization and C5a-stimulated chemotaxis are required for PMN bacterial killing in fibrin gels, and that fMLP inhibits PMN bactericidal activity in fibrin gels. The latter finding is surprising and suggests that in the presence of fibrin fMLP promotes bacterial virulence.     

Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major

Macrophages are the major target cell population of the obligate intracellular parasites LEISHMANIA: Although polymorphonuclear neutrophil granulocytes (PMN) are able to internalize Leishmania promastigotes, these cells have not been considered to date as host cells for the parasites, primarily due to their short life span. In vitro coincubation experiments were conducted to investigate whether Leishmania can modify the spontaneous apoptosis of human PMN. Coincubation of PMN with Leishmania major promastigotes resulted in a significant decrease in the ratio of apoptotic neutrophils as detected by morphological analysis of cell nuclei, TUNEL assay, gel electrophoresis of low m.w. DNA fragments, and annexin V staining. The observed antiapoptotic effect was found to be associated with a significant reduction of caspase-3 activity in PMN. The inhibition of PMN apoptosis depended on viable parasites because killed Leishmania or a lysate of the parasites did not have antiapoptotic effect. L. major did not block, but rather delayed the programmed cell death of neutrophils by approximately 24 h. The antiapoptotic effect of the parasites could not be transferred by the supernatants, despite secretion of IL-8 by PMN upon coculture with L. major. In vivo, intact parasites were found intracellularly in PMN collected from the skin of mice 3 days after s.c. infection. This finding strongly suggests that infection with Leishmania prolongs the survival time of neutrophils also in vivo. These data indicate that Leishmania induce an increased survival of neutrophil granulocytes both in vitro and in vivo.     

Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method

The ultrastructural localization of NADH oxidase, a possible enzyme in the increased oxidative activity of polymorphonuclear leukocytes (PMN) during phagocytosis, was studied. A new cytochemical technique for the localization of H2O2, a product of NADH oxidase activity, was developed. Cerous ions, in the presence of peroxide, form an electron-dense precipitate. Resting and phagocytically stimulated PMN were exposed to cerous ions at pH 7.5 to demonstrate sites of NADH-dependent, cyanide-insensitive H2O2 production. Resting PMN exhibites slight activity on the plasma membrane; phagocytizing PMN had extensive deposits of reaction product localized within the phagosome and on the plasma membrane. Peroxide involvement was demonstrated by the inhibitory effect of catalase on cerium precipitation; the surface localization of the enzyme responsible was confirmed by using nonpenetrating inhibitors of enzymatic activity. A correlative study was performed with an NADH-dependent, tetrazolium-reduction system. As with cerium, formazan deposition on the surface of the cell was NADH dependent, cyanide insensitive, and stimulated by phagocytosis. Superoxide dismutase did not inhibit tetrazolium reduction, as observed cytochemically, indicating direct enzymatic dye reduction without superoxide interposition. These findings, combined with oxygen consumption studies on resting and stimulated PMN in the presence or absence of NADH, indicate that NADH oxidase is a surface enzyme in human PMN. It is internalized during phagocytosis and retains its peroxide-generating capacity within the phagocytic vacuole.     

Effects of leukocyte inhibitory factor (LIF) on neutrophil phagocytosis and bactericidal activity

Human leukocyte inhibitory factor (LIF) is a lymphokine initially defined by its ability to inhibit the random migration of neutrophils. We have recently demonstrated that LIF also potentiates a number of f-met-leu-phe-mediated functions as well as enhancing one Fc receptor-mediated function (antibody-dependent cellular cytotoxicity). In this paper, we have extended our studies involving the effects of LIF on the neutrophil, specifically its effect on phagocytosis and bactericidal activity. We demonstrate that LIF (2 U/ml) potentiates phagocytosis of opsonized heat-killed Staphylococcus aureus (up to 57.2%) and sheep erythrocytes (124.4%) as well as unopsonized latex particles (59.9%). Phagocytosis of opsonized sheep erythrocytes was inhibited by an anti-neutrophil Fc receptor antibody with control PMN but not using the LIF-treated PMN. LIF (1/2 to 1 U) also potentiates the killing of S. aureus by up to 51.6%. Higher concentrations of LIF (greater than or equal to 4 U) inhibits killing. These effects were shown not to be associated with an increase in Fc receptor availability. It is therefore possible that potentiation of these neutrophil activities by LIF may occur either as a result of increased receptor turnover or, more likely, secondary to an increase in nonspecific neutrophil adherence. These studies further support the concept that LIF may have an important role in vivo in inflammation and immunity.     

Interleukin-10 inhibits neutrophil phagocytic and bactericidal activity

Abstract Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells, which is dependent upon the coordinated expression of both pro- and anti-inflammatory cytokines. Interleukin-10 (IL-10) is a recently described cytokine with potent anti-inflammatory properties in vivo and in vitro. In this study we investigated whether IL-10 could directly regulate the ability of neutrophils (PMN) to phagocytose and kill bacteria. Initial studies demonstrated that human recombinant IL-10 (hrIL-10) inhibited the ability of PMN to phagocytose Escherichia coli in vitro. Inhibition of phagocytosis occurred in the absence of changes in CR1 (C3b) or Fc receptor expression, as treatment of PMN with IL-10 failed to induce significant changes in FcγIIR, FcγIIIR or CR1 cell surface expression. However, incubation of PMN with IL-10 resulted in a dose-dependent decrease in CD11b (Mac-1) expression. In addition to effects on PMN phagocytosis, hrIL-10 significantly attenuated PMN microbicidal activity, as bactericidal assays revealed that co-incubation of PMN with hrIL-10 resulted in a marked decrease in killing of phagocytosed bacteria. Furthermore, IL-10 inhibited the production of superoxide from PMA-stimulated PMN, suggesting that the detrimental effects of IL-10 on PMN microbicidal activity were due, in part, to suppression of respiratory burst. In summary, our studies indicate that IL-10 inhibits PMN-dependent phagocytosis and killing of E. coli in vitro, and suggest that this cytokine may impair effective antibacterial host defense in vivo.