Co-occurrence of IgE and IgG autoantibodies in patients with chronic spontaneous urticaria.

Chronic spontaneous urticaria (CSU) pathogenesis shows a complex and still unclear interplay between immunoglobulin (Ig)G- and IgE-mediated autoimmunity, leading to mast cell and basophil degranulation and wheal formation. The objective of this study was to evaluate at the same time IgE- and IgG-reactivity to well recognized and recently reported autoantigens in CSU patients, and to assess the effects of such reactivity on response to the anti-IgE monoclonal antibody omalizumab. Twenty CSU patients underwent omalizumab treatment. Urticaria activity score 7 (UAS7) was recorded at baseline and at different drug administration time-points for categorizing early-, late- or non-responders. At baseline, sera from the 20 patients and from 20 controls were tested for IgE and IgG autoantibodies to high- and low-affinity IgE receptors (FcεRI and FcεRII), tissue factor (TF) and thyroglobulin (TG) by immunoenzymatic methods. Antibody levels were compared with those of controls and analysed according to response. Eighteen patients were omalizumab responders (11 early and seven late), while two were non-responders. More than 50% of patients had contemporary IgE and IgG to at least to one of the four different autoantigens. Late responders showed higher levels of both anti-TF IgE and IgG than early responders (P = 0·011 and P = 0·035, respectively). Twenty-five per cent of patients had levels of anti-FcεRI IgE, exceeding the upper normal limit, suggesting that it could be a novel auto-allergen in CSU. In CSU, there is an autoimmune milieu characterized by the co-existence of IgE and IgG autoantibodies to the same antigen/allergen, particularly in late responders to omalizumab, possibly explaining the slower response.

Involvement of the IgE-basophil system and mild complement activation in haemophilia B with anti-factor IX neutralizing antibodies and anaphylaxis.

INTRODUCTION: Patients with haemophilia B who develop factor IX (FIX) neutralizing antibodies (inhibitors) after FIX infusion are at high risk of hypersensitivity reactions upon FIX re-exposure, but the underlying mechanisms are incompletely understood. AIM: To investigate biomechanisms of FIX hypersensitivity. METHODS: A cellular antigen stimulation test (CAST) was employed to evaluate leukotriene C4 (LTC4) release from basophils stimulated by FIX in three treated children with haemophilia B, one of whom developed FIX inhibitor and experienced anaphylaxis following FIX re-exposure. Anti-FIX IgE and IgG antibodies and markers of complement activation (C5b9, C3d and iC3b) were measured in plasma, the last also after FIX infusion. Ten healthy children served as controls. RESULTS: The patient who developed anti-FIX inhibitors and anaphylaxis had a nonsense mutation in FIX gene (p.Arg298Stop) and, compared to controls, had higher plasma levels of specific anti-FIX IgE (2.285 vs 0.084 OD492 nm ), with marked LTC4 release from his FIX-stimulated basophils (519.8 vs 39.9 pg/mL). Further, he had higher plasma levels of anti-FIX IgG of all the four subclasses (total IgG 1.180 vs 0.120 OD492 nm ) with FIX neutralizing activity (1.5 BU); mild complement activation occurred during FIX-induced anaphylaxis (C5b9 increased from 258.5 to 351.1 ng/mL). The same parameters were normal in the two patients who tolerated FIX infusion. CONCLUSION: In the patient with haemophilia B who experienced anaphylaxis after FIX, but not in the patients with haemophilia B who tolerated FIX, the CAST assay showed FIX-induced LTC4 release, which was associated with high plasma levels of specific anti-FIX IgE and IgG antibodies.

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-gamma (IFN-gamma)-enhanced activities of human neutrophils. An 18h culture of neutrophils with IL-10 dose-dependently down-regulated their capacity to produce O(2)- 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 gamma RI, Fc gamma RII, Fc gamma RIII, CR1, CR3 and Fc gamma R- and CR-mediated phagocytosis were not modified by the cytokine. Culture of neutrophils with IFN-gamma (100 U/ml) did not modify their Fc gamma R- and CR-mediated phagocytosis, but significantly up-regulated Fc gamma RI and CR3 membrane expression as well as their oxidative metabolism and antibody-dependent cellular cytotoxicity (ADCC). When IL-10 and IFN-gamma were added simultaneously to neutrophil culture, IL-10 dose-dependently reduced IFN-gamma-induced increase of CR3 expression, O(2)- production (in response to both FMLP and phorbol 12-myristate 13-acetate, or PMA) and ADCC, but did not change Fc gamma RI 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.

IL-10 up-regulates human monocyte phagocytosis in the presence of IL-4 and IFN-gamma.

Interleukin-10 (IL-10), a cytokine produced by type 2 helper T (Th2) cells, inhibits the microbicidal effector function of interferon-gamma (IFN-gamma)-activated macrophages. However, recent observations indicate that IL-10, like IFN-gamma, increases Fc gamma RI expression and Fc gamma R-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 IL-10 caused a significant enhancement of their capacity to ingest particles coated with immunoglobulin G (Fc gamma R-mediated ingestion) or with C3b/C3bi fragments of the complement system (CR1/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 > or = 1 U/ml. This up-regulating activity on phagocytosis was completely reversed by anti-IL-10 monoclonal antibody (mAb). As previously reported, IL-10 stimulated Fc gamma RI expression on monocytes but did not induce the expression of Fc gamma RII, Fc gamma RIII, CR1, and CR3. IFN-gamma, like IL-10, up-regulated only Fc gamma RI expression but significantly reduced both Fc gamma R- and CR-mediated ingestion. IL-10 almost completely reversed the IFN-gamma-induced inhibition of both Fc gamma R- 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 Fc gamma Rs and also inhibited Fc gamma R-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 Fc gamma R expression but also completely reversed the IL-4-induced suppression of Fc gamma R-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-IL-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.(ABSTRACT TRUNCATED AT 400 WORDS)

Abnormal neutrophil chemotaxis in bone marrow transplant patients correlates with impaired 31D8 monoclonal antibody binding.

BACKGROUND: 31D8 monoclonal antibody (mAb) has been shown to bind heterogeneously to human neutrophils, identifying subsets of cells which differ in their functional response to chemotactic stimuli. In this study we used 31D8 mAb to determine whether differences in neutrophil subpopulations might explain the long-lasting decreased chemotaxis observed in bone marrow transplant recipients. METHODS: Thirty patients with self-sustaining hematopoiesis 1 to 5 years bone marrow transplantation (BMT) (15 allogeneic and 15 autologous) performed for acute lymphocytic leukemia (ALL, 10 patients) or acute myelogenous leukemia in complete remission (8 patients), Hodgkin's lymphoma (2 patients), chronic myeloid leukemia (8 patients) and severe aplastic anemia (2 patients) were included in the study. Neutrophil chemotaxis was evaluated using a modified Boyden chamber assay and 31D8 binding was determined by indirect immunofluorescence and cytofluorimetric analysis. RESULTS: Neutrophil chemotaxis was significantly impaired in the BMT group with respect to controls. The chemotactic defect strikingly correlated with autologous BMT and, in particular, with ALL as the pre-existing disease. No differences between patients and controls were observed in the percentage of 31D8 bright and dull neutrophils. However, when mean fluorescence intensity (MFI) was analyzed as a relative measure of 31D8 antigen expression on the overall neutrophil population, a significant decrease was observed in neutrophils from BMT patients with respect to controls. As for chemotaxis, the impairment of 31D8 binding was more evident in autologous BMT and strikingly correlated with ALL as the pre-existing disease regardless of age, sex and time since BMT. Moreover, a significant positive correlation between impaired chemotaxis and decreased 31D8 binding was found in our patients. CONCLUSIONS: These findings suggest that the decreased neutrophil chemotaxis observed in some BMT patients may be due in part to circulating 31D8 dull neutrophils, although the causes for the decreased 31D8 binding and for the quite pronounced neutrophil defect in ALL patients remain unknown.

Fc receptors expression and function in mononuclear phagocytes from AIDS patients: modulation by IFN-gamma.

Fc-receptor (FcR)-mediated phagocytosis and FcR (FcRI, FcRII and FcRIII) membrane expression was studied on freshly separated and cultured monocytes (Mo) from 20 AIDS patients and 20 healthy controls. Both Mo and Mo-derived macrophages from AIDS patients presented a significant defect in their capacity to ingest IgG-coated erythrocytes (EA) compared to control cells. This functional defect did not depend on a decline in the number of FcR+ cells or on a decrease in the expression of FcR on their surface. In fact, the percentages of phagocytes reacting with anti-FcRI MoAb (32.2) or anti-FcRII MoAb (IV.3) were similar for controls and AIDS patients, while the percentage of FcRIII-positive Mo (MoAb 3G8) was higher in the AIDS population than in controls, though this difference was not seen on cultured Mo. The level of FcRI expression, evaluated as mean fluorescence intensity (MFI), was higher on freshly separated Mo from AIDS patients than from controls but this difference disappeared also with differentiation of Mo to Mo-derived macrophages in vitro. Parallel analysis of FcRII and FcRIII on phagocytes revealed no differences in the MFI between the AIDS and control groups. Some observations suggested that this functional defect might be secondary to phagocyte priming by circulating IFN-gamma: (1) in vitro stimulation of Mo with hrIFN-gamma, which increased FcRI expression, actually reduced phagocytosis of IgG-coated particles; and (2) IFN-gamma concentrations were increased in AIDS patients' plasma. In spite of these findings, no significant correlation was found between plasma IFN-gamma concentrations and FcR-mediated ingestion in AIDS patients, making the hypothesis uncertain. Even if the basis for the impaired FcR-mediated phagocytosis in AIDS patients remains unclear, this functional defect may have a role in the immunopathogenesis of AIDS, constituting a component cause of the immunodeficiency.

Immunomodulation of polymorphonuclear leukocytes by D53 Immucytal and its constitutive fractions.

D53 (Immucytal) is a compositive vaccine made of immunogenic ribosomes extracted from 4 bacterial species (Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pyogenes and Streptococcus pneumoniae) associated with a membrane proteoglycan from a non-encapsulated strain of Klebsiella pneumoniae (MPG-Kp). In this work we have studied the effect of the compound on human polymorphonuclear leukocyte (PMN) function "in vitro". We have demonstrated that D53 was able to significantly increase Fc- receptor dependent phagocytosis without modify the C3-receptor dependent activity. Furthermore D53 enhanced the oxidative metabolism (evaluated by chemiluminescence) both using cells in resting conditions or after stimulation with phagocytable or soluble stimuli. On the contrary D53 caused a dose-dependent inhibition of PMN migration toward different chemoattractants. Using the two constitutive fractions of the compound (ribosomes and proteoglycans) we have observed that the MPG-Kp component was mainly responsible for the modulating activity of the drug on human PMNs.