Increased expression of IgG Fc receptor type I on neutrophils and monocytes from HIV-infected subjects.

Interferon-gamma (IFN-gamma) induces de novo expression of IgG Fc receptor type I (FcRI) on neutrophils and significantly raises the level of these receptors on monocytes. Since increased concentrations of IFN-gamma have been observed in sera from patients with HIV infection, FcRI expression might also be increased on these subjects' phagocytes. FcRI expression was assessed by indirect immunofluorescence staining of phagocytes in whole blood from 40 healthy controls and 55 HIV+ subjects, 24 belonging to CDC class III and 31 to CDC class IV; 42 were intravenous drug abusers (IVDA) and 13 were homosexual men. Plasma levels of IFN-gamma were measured using a modified immunoradiometric assay. The mean linear fluorescence intensity, used as a relative measure of receptor expression, was significantly higher on unseparated neutrophils from HIV+ subjects in CDC classes III (P < 0.001) and IV (P < 0.0001) than from controls. Similar changes in FcRI expression were observed on monocytes from HIV+ subjects. While no differences were observed between IVDA and homosexual HIV+ patients, there was a significant association between FcRI expression and the patients' CDC stage, those in class IV having the highest FcRI levels. Plasma IFN-gamma concentrations were significantly higher in HIV+ patients than in controls and a positive correlation with the stages of HIV infection was again observed. FcRI expression was also increased on freshly purified neutrophils from five HIV+ patients in CDC class IV but did not increase further after 18 h incubation with IFN-gamma, a treatment that up-regulated FcRI expression on control neutrophils. These data suggest that: (i) FcRI evaluation may be a sensitive marker for the biological activity of IFN-gamma in vivo; (ii) phagocytes from HIV+ subjects are activated in vivo by IFN-gamma, expressing increased levels of FcRI; (iii) these IFN-gamma-activated cells may play a role in the pathogenesis of AIDS.     

Regulation of the expression of Fc gamma receptor on circulating neutrophils and monocytes in Kawasaki disease.

To investigate the regulation of Fc gamma receptor (Fc gamma R) expression on circulating phagocytes in Kawasaki disease (KD), we analysed the expressions of Fc gamma RI, II and III on neutrophils and monocytes in 20 patients with KD, 10 with a bacterial infection (BI), 10 with a viral infection (VI), and 10 healthy controls (HC) using flow cytometric analysis. The KD patients had a significantly higher level of Fc gamma RI expression on neutrophils, but not on monocytes, than the BI, VI and HC patients. Fc gamma RII expression on neutrophils was significantly higher in KD, BI and VI than HC, but there was no significant difference in Fc gamma RII expression among KD, BI and VI. Fc gamma RIII expression on neutrophils in KD was significantly lower than in VI and HC, but was higher on monocytes. A kinetic analysis of Fc gamma R expression in KD demonstrated the expression of Fc gamma RI and II on neutrophils to decline, but no remarkable change was observed in the monocytes, from the subacute phase through the convalescent phase. In addition, Fc gamma RIII expression on neutrophils increased, while Fc gamma RIII expression on monocytes decreased during the time course of KD. Fc gamma R expression in the acute phase of KD is thus characterized by markedly increased expression of Fc gamma RI on neutrophils, followed by a subsequent decrease, and decreased expression of Fc gamma RIII on neutrophils and increased expression of Fc gamma RIII on monocytes followed by a reverse kinetics during the clinical course. These findings are thus considered to reflect the functional up-regulation of neutrophils and monocytes in KD.     

Chimeric IgG4 PR3-ANCA induces selective inflammatory responses from neutrophils through engagement of Fcγ receptors

Anti-proteinase 3 antibodies are implicated in the pathogenesis of small vessel vasculitis. These are primarily immunoglobulin G (IgG), with different subclasses predominating at different stages of disease. However, little is known of their respective roles in pathogenesis. We have previously shown that patient IgG4 was able to induce superoxide release from human neutrophils. To circumvent difficulties in separating the subclasses and additional differences in polyclonal patient antibodies we have generated monoclonal mouse/human IgG1 and IgG4 anti-proteinase 3 antibodies. Using these antibodies we have compared effects of IgG1 and IgG4 on human neutrophils in terms of superoxide release, cytokine production, degranulation and adhesion. Additionally we have investigated the interaction of the subclasses with Fc receptors expressed by the neutrophil. Chimeric antibodies were generated using human constant regions of each subclass and a variable region taken from a monoclonal antibody directed against proteinase 3. Superoxide release from neutrophils was measured by the reduction of ferricytochrome C, degranulation by the conversion of a synthetic colour substrate, cytokine release by interleukin-8 enzyme-linked immunosorbent assay, and adhesion by a flow-based adhesion assay. Fc receptor binding was assessed using blocking antibodies. The IgG4 anti-proteinase 3 was able to induce a dose-dependent release of superoxide, degranulation and adhesion. The antibody was not able to stimulate the secretion of interleukin-8. Fc receptors were essential for neutrophil stimulation and the constitutive Fc receptors were necessary for different stimulatory pathways. The IgG4 anti-proteinase 3 antibodies are able to stimulate neutrophils to undergo a pro-inflammatory response and may play a role in the pathogenesis of small vessel vasculitis.     

Platelet activating factor increases expression of complement receptors on human neutrophils

The phospholipid inflammatory mediator platelet activating factor (PAF) has been shown to stimulate certain functions of polymorphonuclear leukocytes (PMN). However, the effect of PAF on surface complement receptors of PMN has not been described. Using monoclonal antibodies and flow cytometry, we have assessed the effects of PAF on surface expression of membrane receptors for C3bi (CR3) and C3b (CR1) in human PMN. PAF (optimal concentration of 1 x 10(-8) M) increased CR3 190% and CR1 174% compared with unstimulated cells at 37 degrees C, while the PAF analogue lyso-PAF had no stimulatory effect. Both CR3 and CR1 responses to PAF reached maximum levels at 15-30 min. PAF effects were comparable to peak effects induced by LTB4 but less than induced by FMLP. A PAF receptor antagonist, SRI 63-441, blocked the increased complement receptor expression in a dose-dependent manner with maximal inhibition of 80-95% at 5 x 10(-6) M. Extracellular calcium had no effect on CR1 expression but slightly enhanced and EGTA partially inhibited the PAF-induced increase in CR3 expression. Simultaneous incubation with PAF and LTB4 enhanced CR3 and CR1 expression more than either agent alone. These findings indicate that PAF, alone and in combination with LTB4, can induce altered expression of complement receptors on the surface of PMN. This effect may enhance adhesion and phagocytosis by PMN at inflammatory reaction sites.     

The up- and down-modulation of immunoglobulin G Fc receptors and complement receptors on activated human neutrophils depends on the nature of activator.

Human neutrophils were activated with soluble stimuli, formyl-methionyl-leucyl-phenylalanine (fMLP) or ionophore A23187, and with opsonized particles, zymosan or Streptococcus pneumoniae bacteria. Monoclonal antibodies and flow cytometry were used to assess the expression of Fc-gamma receptors (FcRI, FcRII, FcRIII) and complement receptors (CR1, CR3). The role of extracellular calcium and magnesium in the modulation of receptor expression was also examined. The low-level expression of FcRI was not affected by any activator tested. fMLP and A23187 did not alter the expression of FcRII, whereas a significant, Ca(2+)- and Mg(2+)-independent down-modulation was observed upon activation with opsonized particles. All activators clearly decreased the surface expression of FcRIII in the presence of Ca2+ and Mg2+, probably as a consequence of shedding of the phosphatidylinositol-glycan-anchored receptor protein. The removal of calcium and magnesium blocked the shedding of FcRIII caused by soluble stimuli, whereas it retarded but did not abolish the fall in FcRIII expression when cells were incubated with opsonized particles. This fall was likely due to internalization of the receptor molecules while the shedding was blocked. A rapid increase in CR1 and CR3 expression was seen upon activation with soluble stimuli. The change in CR1 expression was independent of extracellular Ca2+ and Mg2+. The increase in CR3 number required an influx of divalent cations. No total up-modulation of complement receptors occurred when neutrophils were activated with opsonized particles. However, the kinetic analysis revealed a temporary up-modulation that was followed by a down-modulation. The results indicate that the expression of both Fc-gamma and complement receptors on human neutrophils is changed upon activation and that the up- and down-modulation of these receptors depends on the nature of activator. We also suggest that in neutrophils the FcRIII down-modulation is the result of both receptor shedding and internalization, while FcRII is down-modulated by receptor internalization.     

Measurement of the binding activity of defined IgG aggregates to macrophage Fc receptors

Small soluble IgG aggregates of defined size were prepared from pooled human IgG by gel filtration chromatography, and examined by analytical ultracentrifugation. Three such fractions, dimer-rich, trimer-rich and 25S aggregate were used to inhibit IgG monomer binding in a study of the influence of aggregation in the binding of human IgG1 to mouse macrophage Fc receptors. Of the polymers tested, IgG in the trimeric form was found to bind with the greatest avidity, being 158 times more active than monomeric IgG, whereas IgG as a larger 25S aggregate had an increased binding activity of 80 times; the avidity of IgG as dimer was increased by a factor of 2 over monomeric IgG. The possible mechanisms involved in achieving enhanced binding are discussed.     

Critical role for complement receptor 3 (CD11b/CD18), but not for Fc receptors, in killing of Streptococcus pyogenes by neutrophils in human immune serum

During phagocytosis, surface receptors on neutrophils interact with pathogens opsonized with complement factor C3b/iC3b and in some cases with antibodies. In human immune sera antibodies directed against surface-bound M proteins mediated killing of Streptococcus pyogenes by neutrophils. Surprisingly, blocking of the Fc receptors had little effect on the killing. In contrast, inhibition of C3b/iC3b generation, or blocking of the major neutrophil iC3b receptor CD11b/CD18, enabled S. pyogenes to grow efficiently in immune sera. Inhibition of CD11b/CD18, but not of CD32, the major neutrophil signaling Fc receptor, prevented Streptococcus-induced NADPH oxidase-dependent respiratory burst, and blocking of C3b/iC3b formation inhibited Streptococcus-induced activation of Cdc42, a small GTPase critically involved in transmitting pro-inflammatory signals to the cytoskeleton. Consequently, ligation of CD11b/CD18 by bacteria-bound iC3b is necessary for inducing a neutrophil response leading to elimination of S. pyogenes in immune human serum.     

Interleukin‐10 plays a key role in the modulation of neutrophils recruitment and lung inflammation during infection by Streptococcus pneumoniae

Streptococcus pneumoniae is a major aetiological agent of pneumonia worldwide, as well as otitis media, sinusitis, meningitis and sepsis. Recent reports have suggested that inflammation of lungs due to S. pneumoniae infection promotes bacterial dissemination and severe disease. However, the contribution of anti-inflammatory molecules to the pathogenesis of S. pneumoniae remains unknown. To elucidate whether the production of the anti-inflammatory cytokine interleukin-10 (IL-10) is beneficial or detrimental for the host during pneumococcal pneumonia, we performed S. pneumoniae infections in mice lacking IL-10 (IL-10^−/− mice). The IL-10^−/− mice showed increased mortality, higher expression of pro-inflammatory cytokines, and an exacerbated recruitment of neutrophils into the lungs after S. pneumoniae infection. However, IL-10^−/− mice showed significantly lower bacterial loads in lungs, spleen, brain and blood, when compared with mice that produced this cytokine. Our results support the notion that production of IL-10 during S. pneumoniae infection modulates the expression of pro-inflammatory cytokines and the infiltration of neutrophils into the lungs. This feature of IL-10 is important to avoid excessive inflammation of tissues and to improve host survival, even though bacterial dissemination is less efficient in the absence of this cytokine.     

Toll-like receptors types 2 and 6 and the apoptotic process in human neutrophils

Introduction: Neutrophils (PMN) apoptosis plays an important role in limiting the last phase of inflammatory processes. It is unknown whether Toll-like receptor (TLR)2 acts independently or together with TLR6 in this process. Materials and Methods: The aim of this study was to estimate the relationship between the expressions of TLR2 and TLR6 and the apoptosis of human neutrophils in physiological conditions. We investigated the influence of recombinant human interleukin (IL)-18 and N-formyl-metionyl-leucyl-phenylalanine (fMLP) on the relationships between these receptors and neutrophil apoptosis. Results: Our results showed that after 4-h incubation, the percentage of apoptotic PMNs significantly increased compared with PMN counts before incubation. The stronger expression of TLR2 on the neutrophils suggests that this receptor contributes more significantly to the induction of PMN apoptosis than does TLR6. We also demonstrated an influence of recombinant human IL-18 (rhIL-18) on the expression of TLR6, whereas this effect was not observed in the expression of TLR2. We observed that both rhIL-18 and fMLP inhibited the apoptosis of PMNs and that rhIL-18 had a stronger effect than fMLP. Conclusions: The obtained results suggest that not only TLR2, but also TLR6 plays an important role in the regulation of the apoptosis of PMNs. Changes in the expression of TLR6 and inhibition of apoptosis of PMNs by rhIL-18 seem to confirm the vital role this receptor and of rhIL-18 in regulating the survival of these cells. These data can be useful in developing methods to regulate PMN apoptosis in conditions associated with their excessive and unfavorable activation.     

The expression of Fc receptors on guinea-pig peritoneal macrophages and neutrophils.

The equilibrium binding of soluble complexes of guinea-pig anti-dinitrophenyl IgG1 or IgG2 and dinitrophenylated bovine serum albumin (DNPBSA) to homologous peritoneal macrophages and neutrophils has been compared. The immunoglobulin receptors on these phagocytes were found to differ in two major respects. Macrophages express specific binding activity for both IgG1 and IgG2 complexes whereas neutrophils possess specificity only for the IgG2 subclass. Furthermore, the number of receptors for IgG2 on macrophages (0.8-1 x 10(6)) is fifty- to seventy-fold greater than the number on neutrophils (1.3-2.6 x 10(4)). The phagocytes also displayed differences in their avidity for soluble IgG2-containing complexes which could either reflect the disparity in receptor densities on their membranes or indicate differences in the structure of their Fc receptors. Inhibition of complex binding by immunoglobulin fragments indicated that, at least, the macrophages and neutrophils recognize the same portion of the IgG2 molecule.     

Fusion of the Fc part of human IgG1 to CD14 enhances its binding to gram-negative bacteria and mediates phagocytosis by Fc receptors of neutrophils

Microbial resistance to antimicrobial drugs is promoting a search for new antimicrobial agents that target highly conservative structures of pathogens. Human CD14 - a known pattern recognition receptor (PRR) which recognizes multiple ligands from different microbes might be a worthy candidate. The aim of our work was to create a CD14/Fc dimer protein and evaluate its whole bacteria binding and opsonizing capabilities. Fusion of CD14 with the fragment crystallisable (Fc) part of human IgG1 could not only lead to an artificial opsonin but the dimerization through the Fc part might also increase its affinity to different ligands. Human CD14 and the Fc part of human IgG1 was fused and expressed in HEK293 cells. A histidine tagged CD14 (CD14/His) was also expressed as control. Using flow cytometry we could prove that CD14/Fc bound to whole Gram-negative bacteria, especially to short lipopolysaccharide (Ra and Re) mutants, and weak interaction was observed between the fusion protein and Listeria monocytogenes. Other Gram-positive bacteria and fungi did not show any association with CD14/Fc. CD14/His showed about 50-times less potent binding to Gram-negative bacteria. CD14/Fc acted as an opsonin and enhanced phagocytosis of these bacteria by neutrophil granulocytes, monocyte-derived macrophages and dendritic cells. Internalization of bacteria was confirmed by trypan blue quenching and confocal microscopy. On neutrophils the Fc part of the fusion protein was recognized by Fc receptors (CD16, CD32), as determined by blocking experiments. CD14/Fc enhanced the killing of bacteria in an ex vivo whole blood assay. Our experiments confirm that PRR/Fc fusion proteins can give a boost to FcR dependent phagocytosis and killing provided the antimicrobial part binds efficiently to microbes.     

Regulation of human T-cell homing receptor expression in cutaneous bacterial infection

We investigated the regulation of T-cell homing receptors in infectious disease by evaluating the cutaneous lymphocyte antigen (CLA) in human leprosy. We found that CLA-positive cells were enriched in the infectious lesions associated with restricting the growth of the pathogen Mycobacterium leprae, as assessed by the clinical course of infection. Moreover, CLA expression on T cells isolated from the peripheral blood of antigen-responsive tuberculoid leprosy patients increased in the presence of M. leprae (2.4-fold median increase; range 0.8-6.1, n = 17), but not in unresponsive lepromatous leprosy patients (1.0-fold median increase; range 0.1-2.2, n = 10; P < 0.005). Mycobacterium leprae specifically up-regulated the skin homing receptor, CLA, but not alpha(4)/beta(7), the intestinal homing receptor, which decreased on T cells of patients with tuberculoid leprosy after antigen stimulation (2.2-fold median decrease; range 1.6-3.4, n = 3). Our data indicate that CLA expression is regulated during the course of leprosy infection and suggest that T-cell responsiveness to a microbial antigen directs antigen-specific T cells to the site of infection.     

The effect of cytokines on the expression and function of Fc receptors for IgG on human myeloid cells

We examined expression and cytotoxic triggering capability of the three Fc receptors for IgG (Fc gamma R) on human monocytes, PMNs and myeloid cell lines after in vitro culture with various cytokines. Fc gamma R expression was evaluated using specific anti-Fc gamma R monoclonal antibodies (mAb). The cytotoxic capability of each Fc gamma R was examined after the effector cells were treated with the recombinant cytokines IFN-gamma. TNF alpha, GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, or IL-6. Hybridoma cell lines (HC) bearing antibody directed to Fc gamma RI (HC 32), Fc gamma RII (HC IV.3) or Fc gamma RIII (HC 3G8) were used as targets, as were chicken erythrocytes (CE) sensitized with heteroantibodies composed of anti-Fc gamma R mAbs (32, IV.3, 3G8) linked to anti-CE antibody. Only IFN-gamma treatment significantly increased Fc gamma R expression and then only Fc gamma RI. IFN-gamma dramatically up-regulated Fc gamma RI expression on all cells tested. However, ADCC was enhanced by treatment with a number of cytokines other than IFN-gamma. GM-CSF, TNF, and IFN-gamma treatment enhanced killing of HC 32 and HC IV.3 by in vitro cultured monocytes. G-CSF treatment enabled PMNs to kill HC through Fc gamma RII, whereas PMN killing of HC through Fc gamma RIII could not be induced by any of the cytokines studied. Although only IFN-gamma treatment increased ADCC of CE by monocytes, GM-CSF treatment as well as IFN-gamma treatment augmented ADCC of CE by PMNs. In addition to IFN-gamma treatment, IL-6 treatment enabled U937 cells to lyse CE. Whereas IFN-gamma-treated U937 cells killed CE through both Fc gamma RI and Fc gamma RII, IL-6-treated U937 cells killed CE only through Fc gamma RI. In addition to IFN-gamma treatment, G-CSF treatment enabled HL-60 cells to lyse CE through both Fc gamma RI and Fc gamma RII. These results demonstrate that although IFN-gamma appears unique in regulating Fc gamma R expression on myeloid cells, cytokines other than IFN-gamma affect ADCC by these cells in a receptor-specific manner.     

The identification of Fc and C3 receptors on human neutrophils.

Electroimmunoassay is a simple, rapid and accurate method for quantitating the serum proteins. By use of glutaraldehyde, intermolecular cross-linkage of immunoglobulins to albumin was effected. The conjugation resulted in increased electrophoretic mobility of the immunoglobulins without perceptible change in antigenic determinants. With a Tris-EDTA-boric acid buffer system, the cross-linked immunoglobulins migrated anodically in an electrical field.Six serum proteins from each of 20 samples were quantitated by electroimmunoassay, and the results correlated with values obtained by radial immunodiffusion.     

Increased expression of human monocyte HLA-DR antigens and Fc gamma receptors in response to human interferon in vivo

The expression of class II MHC encoded antigens (HLA-DR) and Fc gamma receptors by peripheral blood monocytes from untreated patients with small cell carcinoma of the bronchus was compared with that of normal donors. Fc gamma receptor expression was found to be elevated in these patients in comparison with normal. In contrast HLA-DR antigen expression by patients' monocytes was somewhat depressed in comparison with normal. Continuous intravenous infusion of a total of 400 megaunits/m2 of human lymphoblastoid interferon-alpha (IFN-alpha) over a 5 day period markedly increased both monocyte HLA-DR antigen expression and Fc gamma receptor expression in comparison with that of untreated patients and normal donors. The initial increases declined somewhat but were still evident after 3 weeks of intermittent intramuscular IFN-alpha therapy.     

Chlamydia psittaci inhibits apoptosis of human neutrophils by activating P2X7 receptor expression

This study tested the hypothesis that Chlamydia psittaci (C. psittaci) survives and multiplies in human neutrophils by activating P2X7, a nonselective cationic channel receptor expressed constitutively on the surface of these cells. Findings illustrated that P2X7 receptor expression was enhanced in C. psittaci-infected neutrophils. C. psittaci was able to inhibite spontaneous apoptosis of neutrophils through mitochondrial-induced ATP release and IL-8 production. Importantly, inhibiting ATP activation of the P2X7 receptor with AZ10606120 promotes apoptosis, while stimulating P2X7 receptor expression with BzATP delayed spontaneous apoptosis of human neutrophils, suggesting that C. psittaci inhibits apoptosis of human neutrophils by activating P2X7 receptor. This study reveals new insights into the survival advantages of the latent persistent state of C. psittaci and the mechanism by which it evades the innate immune response.