Francesca Minonzio

The neuropeptide α-MSH has specific receptors on neutrophils and reduces chemotaxis in vitro

Abstract The proopiomelanocortin-derived peptide α-melanocyte stimulating hormone (α-MSH) has potent anti-inflammatory effects in all animal models of inflammation against which it has been tested. Understanding of the mechanism by which this occurs is incomplete, although there is recent evidence for α-MSH receptors in murine and human macrophages and for modulation of production of proinflammatory cytokines and related mediators by α-MSH. Because of the prominence of neutrophils in early stages of inflammatory reactions where α-MSH is effective, we examined human neutrophils for evidence of mRNA for α-MSH receptors and for inhibition of neutrophil chemotaxis. There was accumulation of mRNA for melanocortin receptor 1 (MC1) in RT PCR product from neutrophils stimulated with interferon and LPS. In subsequent studies α-MSH inhibited migration of neutrophils from most normal volunteers when the cells were placed in FMLP or IL-8 gradients. The inhibition by α-MSH could be traced to alterations in cAMP in neutrophils. The presence of α-MSH receptor message in neutrophils is consistent with the established anti-inflammatory effects of the peptide. Direct inhibition of neutrophil chemotaxis likely contributes to the anti-inflammatory activity of α-MSH.

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.

Effect of adalimumab on neutrophil function in patients with rheumatoid arthritis

Neutrophils are known to be targets for the biological activity of tumour necrosis factor (TNF)-α in the pathogenensis of rheumatoid arthritis (RA). Therefore, these cells may be among the targets of anti-TNF-α therapy. In this study we evaluated the effect of therapy with adalimumab (a fully human anti-TNF-α mAb; dosage: 40 mg subcutaneously every other week) on certain phenotypic and functional aspects of neutrophils obtained from 10 selected patients with RA and 20 healthy control individuals. Peripheral blood neutrophils were obtained at baseline and during anti-TNF-α therapy (2, 6 and 12 weeks after the first administration of adalimumab). All patients had been receiving a stable regimen of hydroxychloroquine, methotrexate and prednisone for at least 3 months before and during the study. Baseline neutrophil chemotaxis was significantly decreased in RA patients when compared with control individuals (P < 0.001). Two weeks after the first administration of adalimumab, chemotactic activity was completely restored, with no differences noted between patients and control individuals; these normal values were confirmed 6 and 12 weeks after the start of anti-TNF-α therapy. Phagocytic activity and CD11b membrane expression on neutrophils were similar between RA patients and control individuals; no modifications were observed during TNF-α neutralization. The production of reactive oxygen species, both in resting and PMA (phorbol 12-myristate 13-acetate)-stimulated cells, was significantly higher in RA patients at baseline (P < 0.05) and was unmodified by anti-TNF-α mAb. Finally, we showed that the activation antigen CD69, which was absent on control neutrophils, was significantly expressed on neutrophils from RA patients at baseline (P < 0.001, versus control individuals); however, the molecule was barely detectable on cells obtained from RA patients during adalimumab therapy. Because CD69 potentially plays a role in the pathogenesis of arthritis, our findings suggest that neutrophils are among the targets of anti-TNF-α activity in RA and may provide an insight into a new and interesting mechanism of action of anti-TNF-α mAbs in the control of inflammatory arthritis.

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.

Severity assessment of healthcare-associated pneumonia and pneumonia in immunosuppression

The study compares the ability of the PSI (pneumonia severity index), CURB-65 (confusion, urea >7 mol·L−1, respiratory rate ≥30 breaths·min−1, blood pressure <90 mmHg systolic or ≤60 mmHg diastolic, and age ≥65 yrs), CURB and CRB-65 scales and the Severe Community-Acquired Pneumonia (SCAP) score to predict 30-day mortality in healthcare-associated pneumonia (HCAP) patients, and analyses differences in the demographics, aetiology and outcomes of community-acquired pneumonia (CAP), HCAP and pneumonia in immunocompromised patients. 629 consecutive patients admitted to a tertiary care university hospital were prospectively categorised as having CAP (n=322) or HCAP (n=307), and the HCAP patients were further sub-divided into those who were immunocompromised (n=219) or immunocompetent (n=88). The 30-day mortality rate was 9.0% in the CAP group and 24.1% in the HCAP group. In the HCAP group, the PSI and SCAP scores had similar prognostic power (area under the curve (AUC) of 0.68 and 0.67, respectively) and performed better than the CURB-65 score (AUC ≤0.62). Among the immunocompetent HCAP patients, the PSI and CURB-65 scores were more sensitive than the others at every threshold, whereas SCAP was more specific than both of these. In the immunocompromised group, the PSI was highly sensitive but poorly specific at all thresholds. Our results suggest that prognostic tools should be designed for subsets of HCAP patients.

Plasma ACTH-response to the corticotropin releasing factor in patients with Cushing's disease. Comparison with the lysine-vasopressin test

In three female patients with Cushings disease, 100 micrograms of synthetic ovine corticotropin releasing factor (CRF) were administered before surgery and 1 week after transsphenoidal microadenomectomy. In these patients a test with lysine-vasopressin (LVP), 10 U intramuscularly, was also performed before and after pituitary surgery. Before surgery, both stimuli induced a clear increase in plasma ACTH and cortisol in all patients; the response of ACTH to CRF was of greater magnitude. Postoperatively, the responses were virtually absent in two patients, but were still present in the third one in whom the removal of adenoma had been partially unsuccessful. The CRF test was also performed in a female patient with Cushings syndrome due to adrenal adenoma; in this patient no responses of plasma ACTH and cortisol to CRF were recorded. This paper demonstrates that pituitary microadenomas causing Cushings disease may retain the ability to respond to CRF; this stimulus may be useful in the differential diagnosis between ACTH-dependent and independent Cushings syndrome; the lack of response after microadenomectomy indicates successful removal of the tumor. CRF is more potent than LVP in releasing ACTH at the doses employed.

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.

Immunopharmacological Activity of Cefodizime in Young and Elderly Subjects: In Vitro and Ex Vivo Studies

SummaryThe biological response modifying activities of cefodizime (CDZ), a new third-generation cephalosporin, were investigatedin vitro andex vivo. In vitro investigations using cells isolated from the blood of young healthy donors showed no stimulating activity of CDZ on peripheral blood lymphocytes, natural killer cell activity, IL-1 production by adherent mononuclear cells, PMN chemiluminescence or PMN chemotaxis. A slight but statistically insignificant increase in PMN phagocytosis and phagocytic index was observed in the same population. IL-1 production was increased in three subjects with low resting state values. In a controlledex vivo study, 20 health elderly subjects selected on the basis of depressed phagocytic function were treated with CDZ 1 g i.m. b.i.d. or placebo for eight days. PMN function was determined at baseline and on the day after the last dose. In the CDZ group a significant increase in both phagocytosis and phagocytic index was found, while there were no changes in the placebo group. In conclusion, CDZ restored depressed PMN phagocytic function in a population of elderly subjects. Patients with impaired PMN function who require antibiotic treatment may benefit from this activity of CDZ.ZusammenfassungDie immunologischen Wirkungen von Cefodizim (CDZ), einem neuen Cephalosporin, wurden untersucht. In einerin vitro Studie mit Zellen, die aus dem Blut junger, gesunder Spender isoliert wurden, zeigte CDZ keine stimulierenden Wirkungen auf Lymphozyten, NK-Zell-Aktivität, IL-1 Produktion durch haftende mononukleäre Zellen, die Chemilumineszenz und Chemotaxis von polymorphkernigen neutrophilen Granulozyten (PMN). Ein geringer, statistisch nicht signifikanter Anstieg der PMN Phagozytose und des Phagozytose-Index wurde beobachtet. Bei drei Personen mit niedriger IL-1 Produktion in Ruhe konnte ein Anstieg derselben induziert werden. Im Rahmen einer kontrolliertenex vivo Studie erhielten 20 gesunde ältere Versuchspersonen, die aufgrund einer verminderten PMN Phagozytose ausgewählt worden waren, CDZ 2 × 1 g i.m. oder Plazebo über einen Zeitraum von acht Tagen. Die Funktion der PMN wurde vor Behandlungsbeginn und am Tag nach der letzten Dosis bestimmt. In der CDZ-Gruppe kam es zu einem signifikanten Anstieg der Phagozytose und des Phagozytose-Index, während in der Plazebo-Gruppe keine Veränderungen beobachtet wurden. Schlußfolgerung: CDZ konnte bei älteren gesunden Versuchspersonen eine verminderte Phagozytosetätigkeit der PMN wiederherstellen. Patienten mit gestörter PMN-Funktion, die einer antibiotischen Behandlung bedürfen, könnte diese Eigenschaft von CDZ zugute kommen.