Anna Maria Ongari

IL‐10 up‐regulates human monocyte phagocytosis in the presence of IL‐4 and IFN‐γ

Interleukin‐10 (IL‐10), a cytokine produced by type 2 belper T (Th2) cells, inhibits the microbicidal effector function of interferon‐γ (EFN‐γ)‐activated macrophages. However, recent observations indicate that IL‐10, like IFN‐γ, increases FcγRI expression and FcγR‐mediated cytotoxic activity on human monocytes, suggesting that this cytokine cannot be classified purely as a monocyte deactivator. The present study found that incubation for 40 h of human monocytes or monocyte‐derived macrophages in the presence of EL‐10 caused a significant enhancement of their capacity to ingest particles coated with immunoglobulin G (FcγR‐mediated ingestion) or with C3b/C3bi fragments of the complement system (GR1/CR3‐mediated ingestion). The number of phagocytosing cells (% phagocytosis) and the number of ingested particles per cell (phagocytic index) were both significantly higher after 40‐h incubation of monocytes with IL‐10 concentrations ≥1 U/ml. This up‐regulating activity on phagocytosis was completely reversed by anti‐EL‐10 monoclonal antibody (mAb). As previously reported, ILIO stimulated FcγRI expression on monocytes but did not induce the expression of FcγRII, FcγRIII, CR1, and CR3. IFN‐γ, like EL‐10, up‐regulated only FcγRI expression but significantly reduced both FcγR‐ and CR‐mediated ingestion. IL‐10 almost completely reversed the IFN‐γ‐induced inhibition of both FcγR‐ and CR‐mediated phagocytosis, without concomitant changes in membrane expression of phagocytic receptors. Exposure of monocytes to IL‐4 reduced the membrane expression of all three FcγRs and also inhibited FcγR‐mediated ingestion. On the other hand, IL‐4 up‐regulated both CR3 expression and CR‐mediated ingestion on cultured monocytes. IL‐10 not only neutralized the down‐regulatory effect of IL‐4 on FcγR expression but also completely reversed the IL‐4‐induced suppression of FcγR‐mediated phagocytosis. Exposure of monocytes to a combination of IL‐10 and IL‐4 resulted in a synergistic effect on CR‐mediated ingestion, even though no additive effects were observed on CR membrane expression. Finally, culture of monocytes in medium containing anti‐EL‐10 mAb significantly reduced their capacity to ingest IgG‐ or C3b/C3bi‐coated particles, suggesting a role for endogenously produced IL‐10 in the modulation of phagocytosis by human monocytes. These results demonstrate that IL‐10 is a potent up‐regulator of the phagocytic activity of human mononuclear phagocytes and indicate that this function may be in sensitive balance with the relative concentrations of EL‐10, IL‐4, and IFN‐γ.

Interleukin-10 down-regulates oxidative metabolism and antibody-dependent cellular cytotoxicity of human neutrophils.

The authors investigated the ability of interleukin‐10 (IL‐10) to modulate some constitutive or interferon‐γ (IFN‐γ)‐enhanced activities of human neutrophils. An 18 h culture of neutrophils with IL‐10 dose‐dependently down‐regulated their capacity to produce O2− and lucigenin‐amplified chemiluminescence in response to n‐formyl‐methionyl‐leucylphenyl‐alanine (FMLP). Furthermore, treatment of neutrophils with IL‐10 decreased in a dose‐dependent fashion, their capacity to lyse antibody‐coated sheep erythrocytes. Membrane expression of FcγRI, FcγRII, FcγRIII, CR1, CR3 and FcγR‐ and CR‐mediated phagocytosis were not modified by the cytokine. Culture of neutrophils with IFN‐γ (100 U/ml) did not modify their FcγR‐ and CR‐mediated phagocytosis, but significantly up‐regulated FcγRI and CR3 membrane expression as well as their oxidative metabolism and antibody‐dependent cellular cytotoxicity (ADCC). When IL‐10 and IFN‐γ were added simultaneously to neutrophil culture, IL‐10 dose‐dependently reduced IFN‐γ‐induced increase of CR3 expression, O2− production (in response to both FMLP and phorbol 12‐myristate 13‐acetate, or PMA) and ADCC, but did not change FcγRI expression on phagocytes. These results demonstrate that IL‐10 is a significant neutrophil deactivator and provide new information on the role of IL‐10 in the regulation of neutrophil‐mediated inflammatory processes.

Induction of CD69 activation molecule on human neutrophils by GM-CSF, IFN-γ, and IFN-α

Abstract The CD69 glycoprotein is an early activation antigen of T and B lymphocytes but it expression is induced in vitro on cells of most hematopoietic lineages, including neutrophils after stimulation with PMA or fMLP. In this study, we investigated whether CD69 expression on human neutrophils could be modulated by inflammatory or anti-inflammatory cytokines (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, G-CSF, GM-CSF, TNF-α, TGF-β, IFN-α, IFN-γ). Resting neutrophils from healthy subjects did not express CD69 on the cell surface; moreover, a preformed intracellular pool of CD69 was not evident in these cells. CD69 was barely detectable on these cells after overnight incubation in medium while overnight incubation with GM-CSF, IFN-γ or IFN-α significantly induced CD69 expression on neutrophils with GM-CSF appearing to be the most potent inducer. This induction was dependent on a new protein synthesis as it was significantly inhibited by cycloheximide (about 50% inhibition). CD69 cross-linking on GM-CSF-primed neutrophils sinergized with LPS and increased TNF-α production and secretion suggesting a role for CD69-positive neutrophils in the pathogenesis and maintenance of different inflammatory diseases.

A new simplified single-filter assay for "in vitro" evaluation of chemotaxis of 51Cr-labeled polymorphonuclear leukocytes

A new simplified radioassay for measuring polymorphonuclear leukocyte (PMN) chemotaxis is proposed using 51Cr-labeled cells and a single-filter system. The technique offers all the advantages described for the double-filter radioassay and permits a reproducible measurement of random locomotion, chemokinesis and chemotaxis. Moreover the single-filter radioassay utilizes commercially available and disposable chambers gathered in a multichamber apparatus; this makes the method very easy to learn and rapid to perform.

In vitro and ex vivo effect of RU41740 on human polymorphonuclear leukocyte function

We investigated the effect of RU41740, a glycoprotein extracted from Klebsiella pneumoniae and possessing immunomodulating properties, on human neutrophil functions in vitro and ex vivo. Our in vitro results showed that RU41740 increased complement- and Fc receptor-dependent phagocytosis. Moreover, the drug enhanced the oxidative metabolism (assessed by chemiluminescence) both in resting and stimulated cells; in the latter case the RU41740-induced enhancement was observed when neutrophils were stimulated with opsonized particles of N-formyl-methionyl-leucyl-phenylalanine (FMLP) but not when phorbol myristate acetate was used. Using otherwise effective experimental conditions, RU41740 did not affect spontaneous or FMLP-induced neutrophil migration. For the ex vivo experience we tested neutrophils of ten elderly subjects with a previously demonstrated phagocytic defect. These subjects were treated orally with RU41740 at a daily dose of 2 mg for 1 week during the first month, and of 1 mg for 1 week in the second month. In this population, RU41740 was able to restore the impaired phagocytic activity and to induce a significant increase of spontaneous chemiluminescence (CL); stimulated CL was also positively influenced. These effects on neutrophils provide new explanatory bases for the immunostimulatory activity of RU41740.

Treatment with PEG‐interferon and Ribavirin for Chronic Hepatitis C Increases Neutrophil and Monocyte Chemotaxis

The incidence of infections increases during treatment with pegylated interferon (PEG‐IFN) and ribavirin (RBV) for chronic hepatitis C (CHC). Despite a reduction in neutrophil count, there is no clear relationship between infection occurrence and neutropenia. In the present study we investigated whether HCV treatment alters leukocyte function. We studied cell chemotaxis, reactive oxygen species, neutrophil phagocytosis, CR3 expression, and plasma colony stimulating factors (CSF) in 20 healthy subjects and 20 patients with CHC (10 with cirrhosis) at baseline, during antiviral treatment (at 4, 12, 24 weeks), and 12 weeks after discontinuation. Our results demonstrate that neutrophil chemotaxis and oxidative burst significantly increased during treatment and returned to baseline at the end of therapy. CR3 neutrophil expression was enhanced in baseline CHC compared to controls but did not change during antiviral treatment. Chemotaxis, oxidative burst, phagocytosis, and CSF levels did not differ significantly between patients before treatment and control subjects or among CHC cases according to the presence of cirrhosis in either cell subpopulation. In conclusion, the innate immune cell activity is enhanced in patients with CHC during antiviral treatment and returns to normal after its discontinuation thus possibly playing a role in their susceptibility to infections.

The Protein Kinase C Inhibitor Aeb071 (Sotrastaurin) Modulates Migration and Superoxide Anion Production by Human Neutrophils In Vitro

We examined the effect of the protein kinase C-selective inhibitor AEB071 (sotrastaurin) on neutrophil functions in vitro. Pre-incubation with AEB071 at concentrations similar to those reached during in vivo therapy significantly reduced cell capacity to migrate toward three different chemo-attractants and to produce superoxide anions (O2) in response to phorbol myristate acetate (PMA) or to iV-formyl-methionyl-leucyl-phenylalanine (fMLP). AEB071 also significantly inhibited the O−2 “overproduction induced by fMLP in neutrophils primed with tumor necrosis factor alpha (TNF-α) or granulocyte/macrophage-colony stimulating factor (GM-CSF). This inhibition was not linked to fMLP-receptor down-regulation since the drug had no effect on either fMLP-receptors or fMLP-induced CD11b membrane expression. When the activity of AEB071 was compared to that of the conventional protein kinase C (PKC) inhibitor Gö6850 (which, like sotrastaurin, inhibits classical and novel PKC isoforms), Gö6976 (an inhibitor of α and β PKC isoforms) and rottlerin (a prevailing δ PKC isoform inhibitor), AEB071 at an equimolar concentration of 3 μM (close to the maximum drug concentration reached in patients treated with AEB071) caused significantly more inhibition on both chemotactic response and superoxide production. These in vitro findings suggest that neutrophils may offer a cellular target for AEB071 activity in vivo.

Effect of Efalizumab on Neutrophil and Monocyte Functions in Patients with Psoriasis

We evaluated the effect of efalizumab on neutrophil and monocyte functions. The in vitro preincubation with efalizumab concentrations similar to those reached during in vivo therapy almost completely saturated CD11a binding sites without affecting the membrane expression of CD11b, CD128a or CD128b. There was a significant reduction in the chemotactic activity of the pre-treated cells toward three different chemo-attractants, whereas their phagocytic capacity and production of oxygen radicals remained unchanged. One month after the administration of efalizumab to five patients with psoriasis (T1) circulating neutrophil counts increased by 34% from pre-therapy (T0) with no change in the number of monocytes. In the same patients the CD11a binding sites on phagocytes were >90% saturated, and there was also a significant down-modulation on neutrophils (44% of T0) and monocytes (63% of T0). In line with in vitro results, efalizumab treatment caused a significant deficiency in the chemotactic properties of neutrophils and monocytes, but no changes in phagocytosis, oxidative burst, production of pro-inflammatory cytokines or the membrane expression of CD11b, CD128a and CD128b. Our findings suggest that neutrophils and monocytes may be among the targets of efalizumab activity in patients with psoriasis.

Increased expression of C3b and C3bi receptors on human neutrophils and monocytes induced by a glycoprotein extract from Klebsiella pneumoniae (RU41740)

RU41740 is a glycoprotein extract from Klebsiella pneumoniae with immunomodulating properties under different experimental conditions. In particular the compound is able to stimulate several functions of human phagocytes in vitro and ex vivo. Using monoclonal antibodies and flow cytometry, in this work we assessed the effect of RU41740 on surface expression of receptors for C3b (CR1) and C3bi (CR3) in human phagocytic cells in vitro. The incubation of whole blood with varying RU41740 concentrations led to a dose-dependent increase in surface expression of CR1 and CR3 on both neutrophils and monocytes when compared with control samples incubated in buffer alone. The maximal drug-induced enhancement of complement receptors was: 291% +/- 13.4% for CR1 and 265% +/- 8.5% for CR3 in neutrophils; 117% +/- 4.5% for CR1 and 98% +/- 4.1% for CR3 in monocytes. These peak effects were observed using RU41740 at a final concentration of 10 micrograms/ml and were similar to those induced by optimal concentrations of the activating compound N-formyl-methionyl-leucyl-phenylalanine (10(-7)M). Polymyxin B did not modify the RU41740-induced enhancement of CR1 and CR3 expression on phagocytes, suggesting no role for endotoxin in this activity. These results define, at least in part, the mechanism of action of RU41740 on human phagocytes in vitro and could be relevant to in vivo events during RU41740 treatment.

Membrane Expression and Function of Complement Receptors CR1 and CR3 on Neutrophils from HIV‐Infected Subjects: Modulation by rTNF‐α and rGM‐CSF

We evaluated membrane expression and function of complement receptors CR1 and CR3 on neutrophils from 27 HIV‐positive (HIV+) subjects (14 in the CDC class III and 13 class IV) as well as their modulation in vitro by recombinant tumour necrosis factor‐α (rTNF‐α) and granulocyte‐macrophage colony stimulating factor (rGM‐CSF). While CR1 was expressed at similar levels on neutrophils from controls and HIV+ subjects. CR3 expression was significantly higher in CDC class IV subjects than in healthy controls. CR1 and CR3 expression was significantly increased after treatment of neutrophils with both cytokines, without differences between controls and HIV+ subjects. Similarly, the superoxide anion (O2) production in response to C3‐coated zymosan (C3zy) was significantly enhanced on neutrophils from CDC class IV subjects when compared with controls. rGM‐CSF and rTNF‐α treatment significantly enhanced the spontaneous as well as C3zy‐stimulated O2 production by neutrophils from controls and CDC class III subjects, and induced an upward trend in the CDC class IV group. These results indicate that the neutrophils of HIV+ patients are preactivated in vivo but they also indicate that these cells may correctly respond to a subsequent particulate stimulus as well as to activating cytokines. Our findings suggest that desensitization or functional exhaustion of complement receptors are not implicated in the abnormalities observed on neutrophils from HIV+ patients.