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

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

The synthetic melanocortin (CKPV)(2) exerts broad anti-inflammatory effects in human neutrophils

Natural melanocortin peptides exert broad effects on the host and they have remarkable therapeutic potential. However, successful use of melanocortins as therapeutic agents depends on the design of molecules that have more stable pharmacological profiles. The synthetic peptide (CKPV)2, based on the C-terminal sequence of a-melanocyte stimulating hormone (a-MSH), has anti-tumor necrosis factor-a (TNF-a) effects in vitro and in vivo and is a promising candidate to treat inflammation. Because neutrophil activity is a major target for anti-inflammatory therapies, we determined whether (CKPV)2 modulates human neutrophil functions in vitro. Incubation of freshly-separated human neutrophils with 10 12\u2013 10 6 M (CKPV)2 significantly inhibited activities relevant to the inflammatory reaction. Neutrophil migration toward the two chemoattractants interleukin 8 (IL-8) and N-formylmethionyl-leucyl-phenylalanine (fMLP) was significantly inhibited by (CKPV)2. (CKPV)2 also inhibited reactive oxygen intermediate (ROI) production induced by phorbol 12-myristate 13-acetate (PMA), but not that induced by fMLP. Because these effects of (CKPV)2 were abolished by the adenylyl cyclase inhibitor 20,50-dideoxyadenosine (ddAdo), they appear to be cAMP-dependent. Finally, the peptide reduced lipopolysaccharide (LPS)-stimulated expression of TNF-a, interleukin-1b (IL-1b), interleukin-8 (IL-8), and intercellular adhesion molecule 1 (ICAM-1), as well as TNF-a protein release in cell supernatants. The data indicate that (CKPV)2 modulates broad cAMP-dependent, anti-inflammatory pathways in human neutrophils

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 (O\u2082\u207b) in response to phorbol myristate acetate (PMA) or to N-formyl-methionyl-leucyl-phenylalanine (fMLP). AEB071 also significantly inhibited the O\u2082\u207b overproduction induced by fMLP in neutrophils primed with tumor necrosis factor alpha (TNF-\u3b1) 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 G\uf66850 (which, like sotrastaurin, inhibits classical and novel PKC isoforms), G\uf66976 (an inhibitor of \u3b1 and \u3b1 PKC isoforms) and rottlerin (a prevailing \u3b4 PKC isoform inhibitor), AEB071 at an equimolar concentration of 3 \u3bcM (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

Induction of CD69 activation molecule on human neutrophils by GM-CSF, IFN-\u3b3, and IFN-\u3b1

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-1\u3b2, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, G-CSF, GM-CSF, TNF-\u3b1, TGF-\u3b2, IFN-\u3b1, IFN-\u3b3). 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-\u3b3 or IFN-\u3b1 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-\u3b1 production and secretion suggesting a role for CD69-positive neutrophils in the pathogenesis and maintenance of different inflammatory diseases

Development of phagocytic function of cultured human monocytes is regulated by cell surface IL-10.

Monocytes differentiating in vitro into macrophages increase their capacity to ingest particles via Fc\u3b3R and CR3. Because human recombinant IL- 10 is a potent up-regulator of phagocytosis in human monocytes, we investigated whether spontaneously produced IL-10 could be a signal for the modulation of phagocytosis by cultured monocytes. We show here that culture of monocytes in the presence of anti-IL-10 mAb completely abolished up- regulation of phagocytosis of both EIgG and EIgMC3bi, suggesting a role for spontaneously produced IL-10 in the modulation of phagocytosis by cultured human monocytes. The inhibition exerted by anti-IL-10 mAb on the development of FcyR-mediated ingestion was dependent on the concomitant inhibition of Fc\u3b3RIII induction in cultured cells. On the other hand, a similar down- regulation of CR3 expression was not involved in the inhibitory effect exerted by anti-IL-10 mAb on the development of CR3-mediated ingestion. Monocytes secreted detectable levels of IL-10 when cultured in medium but the concentrations of IL-10 in the supernatants decreased with length of time in culture, the decrease being completely reversed by anti-IL-10 mAb. In addition, we showed that monocytes expressed immunoreactive IL-10 on their surface and this expression increased during differentiation into macrophages. Whether this IL-10 was bound to specific membrane receptors or it was an integral membrane protein remains to be determined; however, this latter possibility is consistent with our observations that IL-10 did not elute with acid treatment and exogenous IL-10 did not increase surface staining of monocytes. Our data indicate that human mononuclear phagocytes express IL-10 on their membrane and suggest that this cytokine may represent an autocrine signal for the increased phagocytic function observed during differentiation of monocytes into macrophages

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-\u3b3 (IFN-\u3b3)-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 Fc\u3b3RI, Fc\u3b3RII, Fc\u3b3RIII, CR1, CR3 and Fc\u3b3R- and CR-mediated phagocytosis were not modified by the cytokine. Culture of neutrophils with IFN-\u3b3 (100 U/ml) did not modify their Fc\u3b3R- and CR-mediated phagocytosis, but significantly up-regulated Fc\u3b3RI and CR3 membrane expression as well as their oxidative metabolism and antibody-dependent cellular cytotoxicity (ADCC). When IL-10 and IFN-\u3b3 were added simultaneously to neutrophil culture, IL-10 dose-dependently reduced IFN-\u3b3-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 Fc\u3b3RI 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