Phenotypic characterization of macrophages in human term placenta.

Immunohistological techniques have been used to study heterogeneity, frequency and distribution of macrophages and T lymphocytes in chorionic villous mesenchyme, stroma of the amniochorion and decidua of 36 human term placentas obtained at spontaneous normal delivery and by caesarean section, using a panel of monoclonal antibodies (mAb) specific for macrophage phenotypes appearing in acute early (mAb 27E10), late (mAb 25F9) and down-regulatory (mAb RM3/1) stages of inflammation. Significant numbers of macrophages were identified. It could be shown that RM3/1+ macrophage phenotypes which in vitro are strongly dexamethasone-inducible and in vivo appear in down-regulatory stages of inflammatory processes are the major cell population in human term placenta. Macrophages characterized by monoclonal antibodies 27E10 and 25F9, as well as CD4+ and CD8+ cells, were distributed sparsely or were completely absent. The finding of anti-inflammatory macrophage phenotypes to be the predominant mononuclear cell population in human term placenta provides support for a mechanism whereby placenta functions as an active immunosuppressive biological barrier between mother and fetus.     

Murine peritoneal macrophage gangliosides inhibit lymphocyte proliferation

Gangliosides have been shown to act as immunoregulatory agents by altering proliferative responses of lymphocytes to both antigens and mitogens. Most early studies have utilized brain gangliosides and have required high concentrations. The role of endogenous gangliosides from macrophages has remained unexplored. In this study, thioglycolate-elicited murine peritoneal macrophage gangliosides were purified and added to cultures of murine lymphocytes. Nanogram amounts caused a profound inhibition of LPS-induced DNA synthesis of splenocytes and of purified B lymphocytes, without demonstrable cellular toxicity. No effect was seen using asialo-GM1. This effect was present across a wide range of lipopolysaccharide (LPS) doses. Nanogram amounts of macrophage gangliosides also inhibited concanavalin A (ConA)-mediated lymphocyte proliferation. Inhibition of LPS-induced mitogenesis was present even if gangliosides were removed from the extracellular environment after 15-60 min of incubation prior to the addition of LPS. This inhibition was reversible with incubation of ganglioside pre-treated lymphocytes in medium containing serum. These inhibitory properties of macrophage gangliosides are distinct from those found in studies using brain gangliosides, and support a potential role for macrophage gangliosides as negative modulators of lymphocyte proliferation.     

Pattern of cytokine receptors expressed by human dendritic cells migrated from dermal explants.

Different reasons account for the lack of information about the expression of cytokine receptors on human dendritic cells (DC): (a) DC are a trace population; (b) the proteolytic treatment used to isolate DC may alter enzyme-sensitive epitopes; and (c) low numbers of receptors per cell. In the present work the expression of cytokine receptors was analysed by flow cytometry on the population of dermal DC (DDC) that spontaneously migrate from short-term culture dermal explants. DDC obtained after dermal culture were CD1alow, CD1b+, CD1c+, human leucocyte antigen (HLA)-DR+, CD11chigh, CD11b+ and CD32+. The DC lineage was confirmed by ultrastructural analysis. DDC expressed interleukin (IL)-1R type 1 (monoclonal antibody (mAb) hIL-1R1-M1; and 6B5); IL-1R type 2 (mAb hIL-1R2-M22); IL-2R alpha chain (mAb anti-Tac; and hIL-2R-M1) and IL-2R gamma chain (mAb 3B5; and AG14C). DDC did not stain for IL-2R beta chain using four mAbs recognizing two different epitopes of IL-2R beta (mAb 2R-B; Mik-beta 1; and CF1; Mik-beta 3, respectively). DDC were also positive for the cytokine binding chains (alpha chains) of IL-3R (mAb 9F5); IL-4R (mAb hIL-4R-M57; and S456C9); and IL-7R (mAb hIL-7R-M20; and R3434). DDC showed low levels of IL-6R alpha chain (mAb B-F19; B-R6; and B-E23) and its signal transducer gp130 (mAb A2; and B1). DDC strongly expressed interferon-gamma receptor (IFN-gamma R) (mAb GIR-208) and were negative for IL-8R (mAb B-G20; and B-F25). All DDC were highly positive for granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) alpha chain (mAb hGM-CSFR-M1; SC06; SC04, and 8G6) and to a lesser extent for the common beta chain of GM-CSFR, IL-3R and IL-5R (mAb 3D7). On the other hand, reactivity was not found for granulocyte colony-stimulating factor receptor (G-CSFR) (mAb hGCSFR-M1) nor macrophage colony-stimulating factor receptor (M-CSFR) (mAb 7-7A3-17) confirming the DC lineage of DDC. As previously reported for lymphoid DC, DDC expressed tumour necrosis factor receptort (TNFR) 75000 MW (mAb utr-1; hTNFR-M1; and MR2-1) but lacked TNFR 55000 MW (mAb htr-9; MR1-1; and MR1-2). In summary, DDC express receptors for a broad panel of cytokines, even receptors for cytokines whose effects on DC are still unknown (i.e. IL-2R alpha gamma; IL-6R alpha/gp 130; IL-7R alpha gamma).     

The surface phenotypic characterization of lung macrophages in C3H/HeJ mice.

We have investigated the surface phenotypic profile of murine lung macrophages in frozen tissue sections, in single-cell suspensions obtained by endobronchial lavage, and in collagenase digests of parenchymal lung tissue, using a panel of monoclonal antibodies directed against pan macrophage markers and antigens present on distinct lymphoid-associated macrophage subpopulations. Our results indicate that lung macrophages from specific pathogen-free (SPF), lipopolysaccharide (LPS) hyporesponsive C3H/HeJ mice are relatively homogeneous no matter what lung tissue compartment they are obtained from. Their predominant surface phenotype was F4/80weak, M1/70-, MOMA-2+, NLDC-145+, MOMA-1+, SER-4+, which resembles the pattern of expression by lymphoid macrophages rather than representative tissue macrophages such as those found in the peritoneal cavity. These results are consistent with the current paradigm that lung macrophages, like lymphoid macrophages, play an important immunoregulatory role within their microenvironment.     

Quantitative and qualitative analysis of the Fc receptor for IgE (Fc epsilon RII) on human eosinophils

In order to characterize the Fc receptor for IgE (Fc epsilon RII) on human eosinophils, we have compared the binding of human IgE myeloma protein to that of a monoclonal antibody (mAb BB10) directed against a common antigenic determinant of the Fc epsilon RII present on eosinophils, platelets and macrophages. Scatchard analysis of the binding to human eosinophils of the BB10 mAb revealed a linear monophasic binding curve, with a binding affinity of 1.17 x 10(7) M-1 and a number of 10(5) binding sites per cell. Biochemical analysis of the human eosinophil Fc epsilon R, performed by immunosorbent chromatography with either BB10 mAb or IgE, showed under nonreducing conditions a major component of 200 kDa. Under reducing conditions, 3 peptide fragments were obtained, with molecular masses of 45-50, 23 and 15 kDa. Finally, comparative analysis suggested that the Fc epsilon RII of human eosinophils and of a human macrophage cell line (U937) are structurally related and differ from the high-affinity Fc epsilon RI present on basophilic granulocytes.     

Recognition of apoptotic cells by human macrophages: inhibition by a monocyte/macrophage-specific monoclonal antibody

Cells undergoing death by apoptosis are rapidly engulfed by phagocytes in vivo, a highly efficient process which prevents leakage of potentially dangerous intracellular contents from dying cells to neighboring tissue. We have tested a panel of monoclonal antibodies (mAb) specifying a range of human monocyte/macrophage surface antigens for their capacity to inhibit the in vitro recognition of apoptotic cells by human peripheral blood monocyte-derived macrophages. The results identify the antigen defined by the 61D3 mAb, a widely-used marker of monocyte/macrophage lineage cells, as an important mediator of apoptotic cell recognition. In our system, apoptotic, but not viable, cells were recognized by the cultured macrophages and 61D3 was found to inhibit the recognition of all apoptotic cell types tested, including Ca²⁺ ionophore-treated or growth factor-depleted B and T lymphocyte lines, tonsillar germinal center B cells, irradiated peripheral blood lymphocytes and senescing neutrophils. Furthermore, the apoptotic cell recognition pathway specified by 61D3 could be distinguished from that involving the macrophage α_vβ₃ vitronectin receptor which has been shown previously to play an important role in the recognition of apoptotic cells. These results provide further evidence that the mechanisms underlying rapid clearance of apoptotic cells involve multiple phagocyte receptors.     

Investigating the human immunodeficiency virus type 1-infected monocyte-derived macrophage secretome

Mononuclear phagocytes (bone marrow monocyte-derived macrophages, alveolar macrophages, perivascular macrophages, and microglia) are reservoirs and vehicles of dissemination for the human immunodeficiency virus type-1 (HIV-1). How virus alters mononuclear phagocyte immunoregulatory activities to complete its life cycle and influence disease is incompletely understood. In attempts to better understanding the influence of virus on macrophage functions, we used one-dimensional electrophoresis, and liquid chromatography tandem mass spectrometry to analyze the secretome of HIV-1-infected human monocyte-derived macrophages. We identified 110 proteins in culture supernatants of control (uninfected) and virus-infected cells. Differentially expressed cytoskeletal, enzymes, redox, and immunoregulatory protein classes were discovered and validated by Western blot tests. These included, but were not limited to, cystatin C, cystatin B, chitinase 3-like 1 protein, cofilin-1, l-plastin, superoxide dismutase, leukotriene A(4) hydrolase, and alpha-enolase. This study, using a unique proteomics platform, provides novel insights into virus-host cell interactions that likely affect the functional role of macrophages in HIV disease.     

EMR1, the human homolog of F4/80, is an eosinophil-specific receptor

The EGF-TM7 F4/80 is a defining marker of murine macrophage populations. Applying flow cytometric analysis using the newly generated mAb A10, and quantitative real-time PCR, we here report the surprising observation that the human ortholog of F4/80, EGF-like module containing mucin-like hormone receptor (EMR)1, is absent on mononuclear phagocytic cells including monocytes, macrophages, and myeloid dendritic cells. Unexpectedly, we found that EMR1 expression is restricted to eosinophilic granulocytes, where expression is overlapping with the eotaxin receptor CCR3 and the immunoglobulin-like lectin Siglec-8. Absence on other leukocytes, including basophils, implies that EMR1 is a highly specific marker for eosinophils in humans.     

Mechanisms of immunity in typhus infections. VI. Differential opsonizing and neutralizing action of human typhus rickettsia-specific cytophilic antibodies in cultures of human macrophages.

Human peripheral blood monocytes were incubated in vitro for 6 days to allow time for transformation into macrophage-like cells. Cytophilic antibodies in typhus convalescent human serum were demonstrated by addition of Rickettsia mooseri or Rickettsia prowazeki to passively sensitized human peripheral blood monocyte-derived macrophages that were held at 4 degrees C. Rosettes of rickettsiae were found around macrophages sensitized with immune serum but not around macrophages that had been incubated with normal serum. Inhibition of rosette formation occurred if the macrophages were maintained in normal human serum before addition of immune human serum. Rosettes of R. mooseri were also formed around monocytes obtained from an individual infected with R. mooseri. If the antibody-sensitized macrophages were maintained at 34 degrees C, enhanced phagocytosis of R. mooseri or R. prowazeki occurred as compared with macrophages exposed to normal human serum before infection. However, the cytophilic antibody did not significantly inhibit the subsequent growth of R. prowazeki within the macrophages. This is in contrast to results obtained when R. prowazeki was mixed with immune serum before addition to the macrophage. In the latter case, growth of R. prowazeki was largely inhibited. The significance of antibody cytophilic for macrophages in typhus infections is discussed.     

Receptor specific probes for the study of Fc gamma receptor specific function.

Human leukocytes express three distinct families of receptors for the Fc region of IgG (Fc gamma R). We have prepared erythrocytes (E) coated with monoclonal anti-Fc gamma R antibodies for the study of receptor specific phagocytosis using biotin and streptavidin. In this technique, both the E and the monoclonal antibody (mAb) are biotinylated and coupling of the mAb to the E occurs through the use of streptavidin. The same biotin/streptavidin principle was used to prepare E coated with human IgG. Using this technique, receptor specific probes or probes coated with natural ligand (IgG) can be prepared rapidly with the use of small amounts of mAb or IgG. Finally, we have used these receptor specific probes to demonstrate that all three families of Fc gamma R (Fc gamma RI, Fc gamma RII and Fc gamma RIII) expressed on human monocytes and human macrophages are phagocytic receptors.     

Characterization of macrophage subsets regulating murine natural killer cell activity

We examined the relationship of I-A expression by normal murine macrophages to their immunoregulatory role on natural killer cell activity. Macrophages were isolated on the basis of plastic adherence; characterized on the basis of conventional markers such as phagocytic ability, cytoplasmic non-specific esterase activity, surface MAC-1 and F4/80 antigen expression; and then used for functional studies relative to their expression of surface I-A. Two functional macrophage subsets were identified: NK-stimulatory and NK-suppressive subsets. The former function was associated with splenic macrophages, which were predominantly I-A+ as identified with a radioautographic immunolabeling technique; the latter function with peritoneal macrophages which were predominantly I-A-. Loss of macrophage I-A expression in vitro was delayed in the presence of indomethacin and enhanced in the presence of PGE2, indicating that PGE2 down-regulates I-A expression on macrophages. The NK stimulatory function of I-A+ macrophages was attributable to a soluble mediator, identified as IFN-gamma, since the stimulatory ability was abrogated with an anti-IFN-gamma antibody. I-A expression appears to be important for the stimulatory function, since some interference with this function was noted in the presence of anti-I-A antibody. The NK-suppressor function of I-A- macrophages was attributable to the soluble mediator PGE2, since this function was abrogated with indomethacin or anti-PGE2 antibody. These results are relevant to the understanding of normal in vivo immunoregulation by macrophages.     

Association of tissue factor with a γ chain homodimer of the IgE receptor type I in cultured human monocytes

Tissue factor (TF) is a high-affinity receptor for coagulation factors VII (F VII) and VIIa. The F VII/VIIa/TF complex is the major cellular initiator of the extrinsic coagulation cascade. We found that the occupancy of TF by its ligand, F VIIa, is involved with signal transduction and that TF is associated with the γ chain homodimer identified as a component of IgE receptor type I (FcϵRI). When 4-day cultured human monocytes were incubated with F VIIa, several polypeptides, especially a 70-kDa polypeptide, were transiently phosphorylated on tyrosine, residues. These phosphorylation events were inhibited by prior binding of anti-TF monoclonal antibody (mAb) HTF-K14, but not anti-TF mAb HTF-K180 to intact cultured monocytes. HTF-K14 blocked the binding of FVII/Vila to cell surface TF, whereas HTF-K180 did not. Anti-TF immunoprecipitates prepared from 1 % digitonin lysates of cultured human monocytes incorporated phosphate in a γ chain homodimer when incubated with [γ-³²P] ATP. The identity of the TF-associated structures as γ chains was established by immunoblot analysis of anti-TF mAb immunoprecipitates with anti-γ chain mAb. In addition, anti-TF immunoblot analysis showed that TF co-precipitated with anti-γ chain mAb. Our data suggest that γ chains may play an important role in signaling via TF in human monocytes/macrophages.     

Generation of anti-Neu-glycolyl-ganglioside antibodies by immunization with an anti-idiotype monoclonal antibody: A self versus non-self-matter

We have previously generated a murine anti-idiotype (Ab2) monoclonal antibody (mAb) to a murine Ab1 mAb, named P3, which selectively binds Neu-glycolyl (NeuGc)-sialic acid on several monosialo- and disialogangliosides, and also reacts with sulfatides and antigens expressed in human melanoma and breast tumors. This Ab2 mAb, designated as 1E10, induced anti-anti-idiotype antibodies (Ab3) in mice and cancer patients. These Ab3 generated by 1E10 mAb were characterized by bearing P3 mAb idiotopes (Ab3, Id +). But when the specificity of these Ab3 antibodies was tested, no specific humoral response against NeuGc-containing gangliosides was detected in sera from immunized mice. However, hyperimmune sera from melanoma and breast cancer patients vaccinated with this Ab2 mAb were able to react specifically with these gangliosides. The different expression of NeuGc-containing gangliosides in the normal tissues of mice and humans could explain these results. In order to demonstrate these findings in other animal species with a different NeuGc-sialic acid expression, we performed similar studies in monkeys and chickens. In monkeys, as in most mammals, NeuGc-containing gangliosides are self-antigens. In contrast, chickens, like humans, lack the expression of these antigens in normal tissues. Here we report that the antibody response against NeuGc-containing gangliosides induced by immunization with 1E10 mAb was completely different in both species. No specific antibody response against these gangliosides was detected in hyperimmune monkey sera. In contrast, a strong and specific Ab3 response against GM3(NeuGc) and GM2(NeuGc) gangliosides (Ab3, Ag+) was generated in chickens due to the administration of 1E10 mAb.     

Selective stimulation of G-CSF gene expression in macrophages by a stimulatory monoclonal antibody as detected by a luciferase reporter gene assay

We have identified a stimulatory monoclonal antibody (mAb) from autoimmune mice that selectively stimulates granulocyte colony-stimulating factor (G-CSF) gene expression in a mouse macrophage cell line. The induction was observed not only in the cell line, but also in normal peritoneal macrophages. This mAb bound to the monocyte/macrophage cell lines and pre-B leukemia cell lines, but also in normal peritoneal macrophages, whereas it did not bind to normal T and B cells in the spleen or fibroblastic cell lines. It could even bind to a human promyelocytic leukemia cell line, when they were differentiated into monocytic cells. On Western blotting, this mAb mainly recognized an approximately 30-kDa band and it was unique because there have been no reports of membrane-associated proteins with a similar molecular mass found in macrophages. These results suggest that there could be a specific gateway molecule to induce G-CSF in macrophages.     

The p150,95 molecule is a marker of human mononuclear phagocytes: comparison with expression of class II molecules

A new monoclonal antibody (mAb), named 3.9, is described that is specific for the p150,95 molecule, a member of the LFA-1, CR3, p150,95 family of human leukocyte differentiation antigens. The LFA-1 molecule participates in a variety of T cell interactions and the CR3 molecule is the receptor for the complement component iC3b, but little is known about the p150,95 molecule. Here we show that the expression of p150,95 is confined to myeloid cells. mAb 3.9 reacts variably with neutrophils, more strongly with monocytes and is most strongly expressed on tissue macrophages. Using this mAb and others, we have examined the heterogeneity of tissue macrophages. Cells such as Langerhans' cells, dendritic reticulum cells and osteoclasts failed to react with these mAb and thus, probably do not belong to the mononuclear phagocyte lineage. Using a new double-labeling technique, we investigated lymphoid tissue for dendritic cells bearing class II molecules which might function in interactions with T cells. In T cell areas macrophages expressing class II markers were seen but there was no evidence for other types of dendritic or interdigitating cells which expressed class II molecules but not macrophage epitopes. The conclusion from this survey was that the most prominent cell with dendritic morphology found in the T cell areas of lymphoid tissue was a macrophage.     

Modulation of human macrophage functions by gangliosides

In human tumors of neuroectodermal origin cell surface expression of individual gangliosides is either increased or decreased relative to comparable normal cells. We have previously shown that gangliosides shed from melanoma cells can immunomodulate T cell activity. Monocytes/macrophages (m/m) are known to play an important role as accessory and effector cells in immune responses. We therefore investigated the effect of exogenous gangliosides derived from melanoma on m/m functions in vitro. Gangliosides commonly expressed on human melanoma such as GM3, GD3, GM2, and GD2 were investigated, as well as GM1, a major component of human neural tissue. Monocytes were isolated from human peripheral blood mononuclear cell populations, treated with gangliosides in vitro, and evaluated in several functional assays. Treatment of m/m with GM2 and GM3 gave the greatest inhibition of Fc receptor expression. GM1 and GD3 on the other hand most inhibited the production of interleukin-1 (IL-1) by m/m. Production of tumor necrosis factor (TNF) like monocytoxin was not affected by incubation with individual gangliosides. These studies suggest that individual melanoma gangliosides have different regulatory effects on m/m functions.     

GM1, a ganglioside that specifically enhances immunoglobulin production and proliferation in human plasma cells

The effects of gangliosides on human plasma cell responses were studied. Among the various gangliosides tested, only GM1 enhanced immunoglobulin (Ig) production and proliferation in the human plasma cell lines, IM-9 and AF-10, while other gangliosides (GM2, GM3, GD1a, GD1b, GD3, GT1b, and GQ1b) had no effect. Among the various cytokines tested, including interleukin (IL)-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, interferon (IFN)-α and IFN-γ, only IL-6 enhanced Ig production and proliferation in IM-9 and AF-10 cells. However, the enhancement of plasma cell responses by GM1 was specific and was not mediated by IL-6, since GM1 activity was blocked by anti-GM1 monoclonal antibody (mAb), but not by control IgM, anti-IL-6 Ab or the anti-IL-6 receptor mAb, PM1. Conversely, the enhancement by IL-6 was blocked by anti-IL-6 Ab and PM1, but not by anti-GM1 mAb. GM1, but not other gangliosides, also enhanced Ig production and proliferation in freshly separated plasma cells from patients with plasma cell leukemia and in plasma cells generated in vitro. These actions of GM1 were specifically blocked by anti-GM1 mAb, but not by anti-IL-6 Ab or PM1. These results indicate that GM1 may be an important regulator of plasma cell responses.     

Modulation of murine macrophage metabolism by glycolipids: inhibition of LPS-induced metabolism by specific gangliosides

We have investigated the ability of gangliosides isolated from murine brain to modulate macrophage metabolism. LPS elicits a profound increase in glucose consumption by cultured resident macrophages. When highly purified mouse brain gangliosides are added to macrophage cultures, a modest inhibition of baseline glucose utilization occurs. Both disialogangliosides and trisialogangliosides mediate this effect. In the absence of serum, these gangliosides may be toxic to cultured macrophages. GT1b is the only brain ganglioside tested that specifically inhibited LPS-induced macrophage metabolism. Sialic acid appears to be a necessary component of the gangliosides, although it is not sufficient to induce inhibition. Asialoganglioside and free sialic acid have no effect on macrophage metabolism in comparison with intact gangliosides. The inhibition of LPS-induced metabolism may be explained by the ability of LPS to form stable molecular complexes with both disialogangliosides and trisialogangliosides. These experiments also suggest that the ganglioside content of macrophages may influence functional responses of macrophages to exogenous stimuli.