Antibody-mediated erythrolysis and erythrophagocytosis by human monocytes, macrophages and activated macrophages. Evidence for distinction between involvement of high-affinity and low-affinity receptors for IgG by using different erythroid target cells.

Antibody-dependent cellular cytotoxicity (ADCC) and Fc receptor-mediated phagocytosis were determined with human monocytes, monocyte-derived macrophages and activated macrophages, using rabbit IgG-covered sheep red blood cells (EAs) and anti-D-treated human erythrocytes (EAhu) as target cells. Monocyte and macrophage-mediated ADCC were distinguished by different kinetics, monocytes lysing either target more rapidly than macrophages. Macrophage activation by recombinant IFN-gamma (rIFN-gamma) led to a marked increase in ADCC activity against EAhu. This manifested in increased lysis of optimally sensitized target cells, in a sustained lysis of target cells carrying low antibody densities, and as an enhanced resistance to lysis inhibition by competing fluid-phase inhibition by competing fluid-phase IgG. All these effects were less striking when EAs were the target cells. Phagocytosis of EAs by rIFN-gamma-treated cells was strongly suppressed, regardless of the amount of antibody on the target cells, and susceptibility to inhibition by fluid-phase IgG was slightly increased. By comparison, phagocytosis of EAhu was depressed to a lesser degree, and susceptibility to inhibition by fluid-phase IgG was reduced when macrophages were rIFN-gamma treated. These and other experiments suggested that the functional triggering of monocytes and macrophages by EAs involved, at least in part, low-affinity Fc receptors (FcR), whereas EAhu interacted with macrophages via high-affinity FcR. It is shown elsewhere that rIFN-gamma treatment of macrophages increases the expression of high-affinity FcR, but not low-affinity FcR (Jungi, Lerch & Brcic, 1987). Differences in the rIFN-gamma-induced functional alterations assessed with EAhu or with EAs are interpreted therefore as being a consequence of differential involvement of high-affinity FcR and of low-affinity FcR in mediating an effector function. For monitoring rIFN-gamma-induced alterations in the effector capacity EAs are more appropriate targets since up-regulation of high-affinity FcR has a smaller influence on the response to this type of target. Using metabolic inhibitors, ADCC could be dissociated from ingestion suggesting that ADCC is not a post-phagocytic event.     

Surface receptors for IgG and complement on equine alveolar macrophages

Isolated equine alveolar macrophages obtained by bronchopulmonary lavage of four live ponies demonstrated surface receptors for equine IgG, equine IgM, and complement-coated sheep red blood cells, but not equine IgM or complement-coated erythrocytes alone. In addition, demonstration of IgG receptors was found to depend on the level of erythrocyte sensitization and could not be demonstrated by red blood cell rosetting techniques at low levels of sensitization. Demonstration of receptors for equine complement by red cell rosetting techniques required the presence of both IgM antibody and serum derived (complement) components. This is the first such study of receptors on equine alveolar macrophages.     

The interleukin 1beta-converting enzyme, caspase 1, is activated during Shigella flexneri-induced apoptosis in human monocyte-derived macrophages.

Shigella, the etiological agent of bacillary dysentery, rapidly kills human monocyte-derived macrophages in vitro. Wild-type Shigella flexneri, but not a nonvirulent derivative, induced human macrophage apoptosis as determined by morphology and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL). Shigella-mediated macrophage cell death was blocked by the peptide inhibitors of caspases, acetyl-Tyr-Val-Ala-Asp-aldehyde (acetyl-YVAD-CHO) and acetyl-Tyr-Val-Ala-Asp-chloromethylketone (acetyl-YVAD-CMK). Protection from apoptosis by YVAD was observed in monocytes matured in the presence or absence of colony-stimulating factors (CSF) like macrophage-CSF or granulocyte-macrophage-CSF. Furthermore, lipopolysaccharide (LPS) or gamma interferon (IFN-gamma) rendered human macrophages partially resistant to Shigella cytotoxicity. Macrophages stimulated with either LPS or IFN-gamma were also protected by YVAD from Shigella-induced cell death. During Shigella infections of human macrophages, interleukin-1beta (IL-1beta) was cleaved to the mature form. IL-1beta maturation was severely retarded by YVAD, indicating that IL-1beta-converting enzyme (ICE; caspase 1) is activated in Shigella-induced apoptosis. The finding that Shigella induces apoptosis in human macrophages by activating ICE supports the hypothesis that the acute inflammation characteristic of shigellosis is initially triggered by apoptotic macrophages which release mature IL-1beta during programmed cell death.     

Surface markers on human activated T lymphocytes. I. Fc receptors for IgG and IgM

Expression of Fc gamma and Fc mu receptors on human peripheral blood T lymphocytes of two subsets with high (E early rosette forming presumably in vivo activated cells, TEe) and low affinity of E receptors (E late rosette forming presumable resting cells, TEl) was investigated. Different distribution pattern of T gamma and T mu cells in the both examined T cell subsets was found. Thus TEe and TEl subsets have been partially enriched in T mu and T gamma cells, respectively. Furthermore, the results obtained in the PHA-stimulated system have shown that Fc mu receptors do not function as the markers of T cell activation. However, in opposition to this finding Fc gamma receptors may be the early activation markers but only of T cells originally bearing high-affinity E receptors.     

Expression and function of histamine receptors 1 and 2 on human monocyte-derived dendritic cells

BACKGROUND: Histamine is a well-known mediator of inflammatory and allergic reactions and has immunomodulatory capacities. There is increasing evidence that dendritic cells as professional antigen-presenting cells play a major role in the development of allergic reactions. However, a possible link between histamine and dendritic cells has not been investigated thus far. OBJECTIVE: We investigated the effect of histamine on human monocyte-derived dendritic cells (MoDCs). METHODS: Expression of histamine H1 and H2 receptors (H1R and H2R) on MoDCs was assessed by means of RT-PCR and flow cytometry. Functional exploration of these receptors was performed by monitoring the increase of intracellular calcium levels (H1R), cyclic adenosine monophosphate formation (H2R), F-actin polymerization, and IL-12p70 production. RESULTS: MoDCs express both H1R and H2R. Stimulation of dendritic cells with histamine resulted in F-actin polymerization and cyclic adenosine monophosphate production through H2R. Influx of calcium could not be detected after stimulation of dendritic cells with histamine under conditions in which histamine induced calcium influx in monocytes. Histamine and H1R and H2R agonists downregulated IL-12p70 production of prestimulated MoDCs. CONCLUSION: MoDCs express histamine H1 and H2 receptors. Our results indicate chemotactic (F-actin polymerization) and immunomodulatory (inhibition of IL-12p70 production) effects of histamine on MoDCs. Therefore histamine might represent a link between immediate-type hypersensitivity reactions and cellular inflammation in allergic disease (eg, in atopic dermatitis).     

Fc receptors for IgA and other immunoglobulins on resident and activated alveolar macrophages

Alveolar macrophage (AMO) were recovered by bronchoalveolar lavage from mice. We have examined the surface of these cells for receptors of immune effector molecules and compared the normal resident AMO to the 'activated' cell present during extensive lung inflammation caused by the parasite Nippostrongylus brasiliensis 2 days after subcutaneous inoculation. As has been shown previously, the resident alveolar macrophage expresses Fc receptors for IgG (90%) and IgE (17%). We now show that a significant proportion of resident AMO possess an Fc receptor for IgA (14%) and this proportion increases to nearly 30% upon activation of the cell, coincident with an increase in release of plasminogen activator and phagocytic activity. This newly described presence of IgA receptors on AMO represents an important demonstration of potential 'arming' of these cells by the predominant antibody in mucous secretions and direct involvement of the AMO in immune mediated reactions in the lung.     

Cloning of cDNA coding for low-affinity Fc receptors for IgE on human T lymphocytes

Using a cDNA probe corresponding to the membrane-bound formof the B cell receptor for IgE, we have isolated, sequenced,and expressed a cDNA clone which codes for a human T lymphocyteFcR from HUT-78 cells. This T cell FcR cDNA codes for 320 aminoacid residues, and shows high homology to the B cell FcR sequence.The major differences between this T cell and the B cell FcRcDNA sequences are (i) a limited stretch of nucleotides at the5' segment of the coding region which encodes a putative cytoplasmicregion of the FcR molecule and the untranslated 5' end; and(ii) an additional 64 bp segment in the untranslated 3' endcontaining two repeats in tandem with three existing repeatsin the same region. The expression of FcR on T lymphocytes mayreflect involvement of the FcR in regulation of IgE-mediatedresponses. The cytoplasmic difference implies functional activityof the FcR in T lymphocytes that is mechanistically differentfrom the FcR of B lymphocytes.     

The localization of the binding site(s) on human IgG1 for the Fc receptors on homologous monocytes and heterologous mouse macrophages.

Two different methods, a rosette assay and a direct binding assay, have been employed in an examination of the binding of human IgG1 to mouse macrophages. In both cases, inhibition of IgG binding was demonstrated by Fc (CH2 + CH3 domains) and pFc' (CH3 domains) fragments of human IgG. In a homologous system, the binding of 125I-human IgG to human peripheral-blood monocytes was inhibited by the Fc fragment whereas the pFc' fragment was inactive. Scatchard plot analysis of the binding data from both the heterologous and homologous systems allowed association constants and numbers of receptors per cell to be calculated. A more thorough examination of the possible location of IgG Fc-receptor binding sites was made using less orthodox proteolytic cleavage fragments of IgG. The site on human IgG1 responsible for binding to mouse macrophage Fc receptors was confirmed to be within the CH3 domains. Human IgG1 binding to homologous monocytes was shown, using a dimeric C gamma 2 domain fragment, to be via the CH2 domains, and was dependent on the integrity of the covalent interaction between the C gamma 2 domains at the hinge region.     

Expression of high- and low-affinity receptors for C3a on the human mast cell line,HMC-1

The proteolytic cleavage product of complement component 3, (C3a), like C4a and C5a, is a potent anaphylatoxin and induces the production of inflammatory mediators in phagocytes. Notably, mast cells respond to C3a with the release of vasoactive substances, including histamine. We have examined the function and receptor binding of C3a in a human leukemic mast cell line, HMC-1. Similar to chemoattractant agonists in leukocytes, C3a induced rapid cytosolic free calcium concentration increases in HMC-1 cells. EGTA did not diminish this response, indicating that mobilizable Ca²⁺ was from intracellular stores. Receptors for C3a in HMC-1 cells couple in part to Bordetella pertussis toxin-sensitive G-proteins and, therefore, appear to belong to the family of serpentine receptors that require G-proteins for signal transduction. HMC-1 cells express two types of C3a receptors, C3aR1 and C3aR2, that were shown to bind ¹²⁵I-C3a with high-(K_d1 = 2.1–4.8 nM) or low-affinity (K_d2 = 30–150 nM), and both receptors are expressed at high level: 3 × 10⁵–6 × 10⁵ C3aR1/cell and 5 × 10⁵–2.3 × 10⁶ C3aR2/cell. Results from cross-linking experiments with ¹²⁵I-C3a fully agree with the presence of two different classes of C3a receptors in HMC-1 cells. Two membrane proteins with apparent molecular masses of 54–61 kDa (p57) and 86–107 kDa (p97) could be covalently modified with ¹²⁵I-C3a, and this cross-linking was inhibited with an excess of unlabeled C3a. Many of the known agonists for leukocytes including 13 chemokines (IL-8, NAP-2, GROα, ENA-78, IP10, PF4, MCP-1, 2 and 3, RANTES, MIP-1α, MIP-1β and 1309), three neuropeptides (neuropeptide Y, somatostatin and calcitonin), as well as C5a, did not activate HMC-1 cells, indicating that C3a is one of a few protein ligands for which this cell line expresses specific receptors. The apparent selectivity for C3a and the abundant expression of C3a receptors make the HMC-1 cell line an excellent choice for the cloning of the receptor genes.     

Properties of receptors for IgG on human placental cell membranes

The amount of 125I-IgG which bound to membranes isolated from the human placenta was competitively inhibited by the presence of increasing amounts of unlabeled IgG but not by unlabeled albumin. The relationship between membrane-bound and free IgG indicated the presence of membrane receptors with an appreciable affinity for IgG. Incubation of membranes with collagenase or neuraminidase did not results in appreciable reduction of IgG-membrane binding, indicating that neither intact collagen nor sialic acid play an important role in the binding. Placental surface membranes isolated by salt extraction bound 3.79+/- 1.78 (SD) pmol IgG/microgram membrane protein, whereas membranes isolated by differential centrifugation bound only 1.61 +/- 0.24 pmol/microgram (p less than 0.02). The fraction of a preparation of solubilized membranes which bound to an IgG affinity column yielded on polyacrylamide gel electrophoresis three prominent protein bands which had molecular weights of 3.7 X 10(4), 4.5 X 10(4) and 6.0 X 10(4) daltons. These findings are consistent with the existence of a limited number of receptors for IgG on placental membranes, including IgG receptors on the microvillus membrane of the syncytial trophoblast. The latter, in accordance with Brambell's hypothesis, could be of importance in the transplacental transport of maternal IgG.     

Methamphetamine and HIV-1 gp120 effects on lipopolysaccharide stimulated matrix metalloproteinase-9 production by human monocyte-derived macrophages

Monocytes/macrophages are a primary source of human immunodeficiency virus (HIV-1) in the central nervous system (CNS). Macrophages infected with HIV-1 produce a plethora of factors, including matrix metalloproteinase-9 (MMP-9) that may contribute to the development of HIV-1-associated neurocognitive disorders (HAND). MMP-9 plays a pivotal role in the turnover of the extracellular matrix (ECM) and functions to remodel cellular architecture. We have investigated the role of methamphetamine and HIV-1 gp120 in the regulation of lipopolysaccaride (LPS) induced-MMP-9 production in monocyte-derived macrophages (MDM). Here, we show that LPS-induced MMP-9 gene expression and protein secretion are potentiated by incubation with methamphetamine alone and gp120 alone. Further, concomitant incubation with gp120 and methamphetamine potentiated LPS-induced MMP-9 expression and biological activity in MDM. Collectively methamphetamine and gp120 effects on MMPs may modulate remodeling of the extracellular environment enhancing migration of monocytes/macrophages to the CNS.     

FIZZ1 and Ym as tools to discriminate between differentially activated macrophages

Although it is well-established that macrophages can occur in distinct activation states, the molecular characteristics of differentially activated macrophages, and particularly those of alternatively activated macrophages (aaMphi), are still poorly unraveled. Recently, we demonstrated that the expression of FIZZ1 and Ym is induced in aaMphi as compared with classically activated macrophages (caMphi), elicited in vitro or developed in vivo during infection with Trypanosoma brucei brucei. In the present study, we analyzed the expression of FIZZ1 and Ym in caMphi and aaMphi elicited during Trypanosoma congolense infection and show that the use of FIZZ1 and Ym for the identification of aaMphi is not limited to T. b. brucei infection and is independent of the organ sources from which macrophages are obtained. We also demonstrate that FIZZ1 can be used to discriminate between different populations of aaMphi. Furthermore, we studied the effects of various stimuli, and combinations thereof, on the expression of FIZZ1 and Ym in macrophages from different mouse strains and demonstrate that regulation of the expression of FIZZ1 and Ym in macrophages is not dependent on the mouse strain. Finally, we show that these genes can be used to monitor the macrophage activation status without the need to obtain pure macrophage populations.     

In vitro effects of cefotaxime and ceftriaxone on Salmonella typhi within human monocyte-derived macrophages

The main objective of this in vitro study was to assess the effects of cefotaxime and ceftriaxone in killing Salmonella typhi in infected human macrophages. Human monocyte-derived macrophages isolated from peripheral blood of human volunteers were cultured in vitro for macrophage differentiation, and subsequently infected with S. typhi strains (a clinical isolate and a standard strain TA-42) at a cell ratio of 10 : 1. MICs of cefotaxime and ceftriaxone were determined by broth microdilution, and the antibiotics were included in the culture medium at one and five times their MIC values. Samples of cell culture medium taken at 0, 3, 6 and 24 h of incubation were cultured for growth of S. typhi on nutrient agar. Gentamicin (10 mg/L) was included in each well except for the control wells, in order to prevent growth of extracellular S. typhi . Both antibiotics showed good in vitro antibacterial effects against S. typhi strains. There were no statistically significant differences between the extracellular and intracellular effects of antibiotics with regard to elimination of the bacteria. Cefotaxime and ceftriaxone are highly effective against extracellular bacterial growth. The results of our in vitro experiments suggest that cefotaxime and ceftriaxone might also be used clinically against susceptible intracellular pathogens such as S. typhi .     

Up-regulation of receptors for IgA on activated human B lymphocytes

Expression of receptors for the Fc part of IgA (Fc alpha R) by T lymphocytes was recently shown to be up-regulated after activation by T cell mitogens in the absence of IgA. We describe a similar increase on activated human B lymphocytes. Fc alpha R were determined by labelling with human secretory IgA (0.5 mg/ml) and flow cytometry analysis after staining with fluoresceinated goat anti-IgA or goat anti-secretory component F(ab')2 fragments. B-enriched cell suspensions were prepared from peripheral blood or tonsils and activated by Staphylococcus aureus Cowan I, anti-IgM antibodies or E. coli lipopolysaccharide. All three activators increased the percentage of Fc alpha R positive cells although only the former induced significant DNA synthesis. Finally recombinant interleukin 1 (10 nM) and interleukin 2 (10 IU/ml) but not interleukin 4 (300 units/ml) nor low-molecular-weight B cell growth factor induced an increase of Fc alpha R expression. The data show that Fc alpha R can be up-regulated on human B cells in the absence of exposure to IgA.     

Human macrophages simultaneously express membrane-C1q and Fc-receptors for IgG

Membrane C1q (mC1q) of macrophages (MPhi) is a precursor of the IgG-binding serum protein C1q. Thus, mC1q potentially provides one of several Fcgamma binding sites of mature MPhi and we analyzed whether simultaneous expression occurs of established receptors for IgG, FcgammaRI, II, and III, and mC1q during in vitro differentiation of MPhi. Using flow cytometry, immunoprecipitation combined with Western blotting and Northern blot analysis mC1q was hardly detected in freshly isolated blood monocytes, but increasingly in developing monocyte-derived MPhi. Laser scanning fluorescence microscopy confirmed the membrane localization of mC1q. Two-color-staining flow cytometry experiments indicated that mC1q and all three types of FcgammaRs are simultaneously expressed on mature monocyte-derived MPhi. A high correlation was found for the expression of mC1q and FcgammaRs, in particular FcgammaRII, but not mC1q and CD14, another marker of monocytes/MPhi.     

Phagocytosis by receptors for C3b (CR1), iC3b (CR3), and IgG (Fc) on human peritoneal macrophages

Human peritoneal macrophages (HPM) obtained via laparoscopy were examined for the presence and functional capacity of complement and Fc receptors. Between 5 and 20 ml of peritoneal fluid containing 1-2 X 10(6) macrophages/ml was available for each study. Macrophages made up 80-95% of the cells in the fluid. Fc and C3 receptors on HPM were characterized by rosette formation with, and phagocytosis of, IgG- and C3-coated sheep erythrocytes (E). ElgG were bound by 82% and ingested by 63% of HPM, with 4-15 E ingested/HPM. The HPM formed rosettes with EC3b (56%) and EC3bi (71%) but not EC3d,g or EC3d. Antibodies to complement receptors type 1 (CR1) and type 3 (CR3) inhibited rosette formation with EC3b and EC3bi, respectively, indicating that HPM possessed separate and distinct receptors for the C3b and iC3b ligands. In 60% of the samples studied, HPM demonstrated the ability to ingest both EC3b and EC3bi, as well as ElgG. Because of the heterogeneous nature of the cells obtained in peritoneal fluid, due to their progressive change from monocytelike cells into mature macrophages, HPM were separated by 1 g velocity sedimentation into fractions of increasing maturity. They were then examined for phagocytosis via Fc and complement receptors. Fc receptor mediated phagocytosis occurred throughout the monocyte-to-macrophage maturation sequence, while the ability of HPM to ingest via CR1 and CR3 was maturation dependent, with ingestion via CR3 occurring before CR1, in a manner analogous to in vitro differentiation of monocyte-derived macrophages.     

Transforming growth factor-beta1 increases CXCR4 expression, stromal-derived factor-1alpha-stimulated signalling and human immunodeficiency virus-1 entry in human monocyte-derived macrophages

Stromal-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 play crucial roles in leukocyte migration and activation, as well as embryogenesis, angiogenesis, cancer and viral pathogenesis. CXCR4 is one of the major human immunodeficiency virus-1 (HIV-1) coreceptors on macrophages. In many tissues macrophages are one of the predominant cell types infected by HIV-1 and act as a reservoir for persistent infection and viral dissemination. In patients infected by HIV-1, blood and tissue levels of transforming growth factor-beta1 (TGF-beta1) are increased. The purpose of this study was to evaluate the effects of TGF-beta1 on CXCR4 expression and function in primary human monocyte-derived macrophages (MDMs) and rat microglia. TGF-beta1 up-regulated CXCR4 and enhanced SDF-1alpha-stimulated ERK1,2 phosphorylation in these cells. The increased CXCR4 expression in human MDMs resulted in increased susceptibility of the cells to entry by dual-tropic CXCR4-using HIV-1 (D-X4). In contrast, TGF-beta1 failed to increase CCR5 expression or infection by a CCR5-using virus in MDMs. Our data demonstrate that TGF-beta1 enhances macrophage responsiveness to SDF-1alpha stimulation and susceptibility to HIV-1 by selectively increasing expression of CXCR4. The results suggest that increased expression of CXCR4 on macrophages may contribute to the emergence of dual-tropic X4 viral variants at later stages of HIV-1 infection.