Microassay for photometric quantitation of macrophage mediated tumor cytotoxicity using an automated densitometer

A simple and reproducible microassay for the quantitation of macrophage mediated cytotoxicity is described. The method is based on the measurement of absorbance at 630 nm of residual Giemsa stained target cells and effector macrophages using an automated densitometer. Applying this novel method, it was possible to demonstrate time dependent growth characteristics of C3H/MCA and BHK/Py target cell lines. Using C3HeB/FeJ or C3H/HeJ murine effector macrophages and syngeneic transformed fibroblast target cells (C3H/MCA or 3T12), the method was further applied to demonstrate: (1) dose related activation of macrophages by lipopolysaccharide (LPS) and by macrophage activating factor (MAF); (2) synergistic augmentation of MAF-mediated macrophage cytotoxicity by LPS; (3) unresponsiveness of C3H/HeJ macrophages to LPS; and (4) increased cytotoxicity with increasing effector: target cell ratios. Guinea pig peritoneal macrophages were also shown to produce enhanced LPS or MAF-mediated cytotoxicity for C3H/MCA or BHK/Py target cells. The novel method was shown to compare favorably with results obtained by cytotoxic release of [3H]thymidine from prelabeled target cells. The advantages of the method are: (1) the elimination of the need for radioactive materials; (2) the ability to perform quantitation directly in microtiter plates; (3) the relative ease and rapidity in which experiments may be performed and quantitated; (4) its sensitivity and reproducibility; and (5) the ability to simultaneously quantitate and observe the biological events either microscopically or macroscopically.     

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.     

Microassay for the photometric quantitation of cell-associated plasminogen activator using a chromogenic tripeptide substrate

Plasminogen activator (PA), a neutral protease whose primary function is to convert plasminogen to plasmin, is produced by various cells including macrophages, monocytes, endothelial cells, and tumor cells. This study reports the use of the chromogenic tripeptide substrate D-Val-Leu-Lys-p-nitroanilide (S-2251) and an automated microtiter plate reader spectrophotometer for the determination of PA activity in cells and fluids. There was a linear relationship between the time of incubation at 37 degrees C and the square root of the absorbance measured at 405 nm when urokinase was incubated with the substrate in the presence of plasminogen. There was no activity in the absence of plasminogen. The slopes of the lines (square root A 405/time) were directly related to the concentrations of urokinase, up through 0.05 CTA units. Using this assay, we determined the cellular activity of PA in human promyelocytic cells HL-60 (1.33 +/- 0.12 CTA units/mg), human monocytoid cells U937 (1.27 +/- 0.12 CTA units/mg), mouse myeloid leukemia cells RFM/UN (0.70 +/- 0.07 CTA units/mg), freshly isolated normal human monocytes (0.00 +/- 0.00 CTA units/mg), and human monocytes after 7 days in culture (5.66 +/- 0.38 CTA units/mg). There was a variable amount of activity expressed in freshly isolated cells or cell lysates of peritoneal macrophages from normal mice, or mice that had gotten intraperitoneal injections of peptone, thioglycollate, or NaIO4, but after 24 or 48 h of culture, these activities, in general, increased. Using this assay, PA levels in the euglobulin precipitates from human plasma prepared without venous occlusion (0.03 +/- 0.02 CTA units/mg protein) or after 5 min of venous occlusion of the arm (0.18 +/- 0.01 CTA units/mg) were comparable to those reported by others using different assays. Thus, this represents a simple, rapid, accurate assay of PA that should be useful to those in immunology, cell biology, and clinical medicine.     

IgA Fc受体研究进展

IgA Fc受体(FcαRI,CD89)属于免疫受体家族成员,主要表达于单核-巨噬细胞、中性粒细胞、嗜酸性粒细胞和树突状细胞等免疫效应细胞表面。FcαRI能特异的与血清型和分泌型IgA结合,并通过γ链介导一系列免疫反应,包括抗体依赖细胞介导的细胞毒性作用(ADCC)、补体依赖的细胞毒性(CDC)及细胞吞噬作用等。FcαRI是一种双功能受体,在不同的生理条件下可以介导免疫系统的活化和抑制反应。FcαRI在机体免疫防御和在维持系统免疫平衡方面扮演着重要的角色,有望成为治疗人类疾病的新靶点。本文对FcαRI的结构、功能及其应用等研究现状进行综述。     

A new photometric microassay for the quantitation of macrophage Fc receptor function. In vitro enzyme-containing immune complexes clearance (EIC) assay

A new photometric microassay for immune complex (IC) binding to macrophages was developed in a homologous system using glucose oxidase-anti-glucose oxidase complexes (GOAGO) as a model for IC clearance in vitro. Thioglycollate-elicited murine peritoneal cells were incubated with GOAGO solution and then cell-associated glucose oxidase activity was measured after the washing and solubilization of the cell membrane in a microtitre plate. GOAGO binding to macrophages was inhibited in the presence of either IgG or its Fc fragments in a dose-dependent manner, while yeast mannan or IgG Fab fragments had no effect. These results indicated that this binding occurred solely via the Fc receptors on the macrophages. The Fc receptors for GOAGO were eliminated by trypsin digestion of the cells. When the macrophages were cultured with LPS or TPA, GOAGO binding was enhanced compared to that of control, whereas carrageenan treatment suppressed GOAGO binding. The present results suggest that this assay may be of value in the measurement of IC clearance and for studying the expression of Fc receptors on macrophages.     

The effect of oligopeptides on the C3b receptor-mediated functions of rat macrophages

Effects of various substances known to influence macrophage functions on the adherence of C3b-coated sheep erythrocytes (E-C3b) and their phagocytosis by elicited rat peritoneal macrophages (PM) has been studied. Different oligopeptides (OP) having a chemotactic effect, such as tuftsin, angiotensin II (At II) and the OP-fraction of a lymphokine preparation (LK-OP) increased both adherence and phagocytosis of E-C3b in a concentration-dependent fashion. In contrast, neither substances influencing the membrane cation transport nor prostaglandins (PG) and cyclic nucleotides significantly changed the C3b receptor (C3bR)-dependent effector functions of macrophages. Enhanced functions of the C3bR were not associated with an increased capability of PMs for intracellular killing of Candida albicans.     

An extended family of Fc receptor relatives

A surprising number of Fc receptor (FcR) relatives have been recognized recently with the potential capacity to modulate innate and adaptive immune responses. The six human FcR homologs (FcRH1-6), which belong to a phylogenetically conserved gene family, have variable numbers of extracellular immunoglobulin domains of five different subtypes. FcRH immunoregulatory potential is implicated by the presence of consensus tyrosine-based activation or inhibition motifs in their cytoplasmic tails. All but one of these new receptors, FcRH6, are expressed on B cells at different stages in differentiation. Their ligands, function, and prospective roles as diagnostic B cell markers and therapeutic targets are topics of intense interest.     

Activation-induced proteolysis of the cytoplasmic domain of ζ in T cell receptors and Fc receptors

The CD3-T cell receptor (TCR) complex on T cells and the Fcγ receptor type III (FcγRIII)-ζ-γ complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits ζ and γ. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized ζ-p14 heterodimer. ζ-p14 is a component of both CD3-TCR and FcγRIII-ζ-γ. Peptide mapping analysis shows that p14 is structurally related to ζ, suggesting that it is either: (i) derived from ζ proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding ζ express ζ-p14 supports the former possibility. The expression of CD3-TCR complexes including ζ-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of ζ may contribute to receptor modulation or desensitization.     

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.     

C3b acceptors on macrophages: inhibition of Fc gamma-receptor-mediated phagocytosis by acceptor-bound C3b

The binding of nascent human C3b (i.e. the fragment of C3 just after trypsin cleavage) to mouse peritoneal macrophages was demonstrated by immune adherence. Acceptor-bound C3b could be detected longer than 24 h on the cell membrane. The rosette formation and phagocytosis of SRBC coated with anti-SRBC rat IgG was inhibited by preincubation of the cells with C3 and trypsin (15 min, 37 degrees C). However, the phagocytosis of opsonized yeast particles was not influenced by acceptor-bound C3b, proving that C3b-C3b acceptor interaction did not alter the function of C3b-receptors. Acceptor-bound C3b on the macrophages failed to mediate phagocytosis of human 0,Rh+ red cells having C3b-receptors.     

In vitro and in vivo macrophage function can occur independently of SLP-76

Expression of SH2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), a hematopoietic cell-specific adapter protein, is required to couple Syk family tyrosine kinase activation to downstream mediators such as phospholipase C (PLC)-gamma following TCR, platelet collagen receptor and mast cell Fc epsilon R stimulation. In addition to T cells, mast cells and platelets, SLP-76 is expressed in monocytes and macrophages. To determine the role of SLP-76 in Fc gamma R-stimulated signaling pathways in macrophages, we examined cultured bone marrow-derived macrophages (BMM) from SLP-76(-/-) and wild-type mice. In this study, we show that Fc gamma R cross-linking rapidly induces tyrosine phosphorylation of SLP-76 in wild-type BMM. Surprisingly, however, BMM from SLP-76(-/-) mice activate ERK2 and phosphorylate PLC-gamma 2 following Fc gamma R ligation. Furthermore, SLP-76(-/-) BMM display normal Fc gamma R-dependent phagocytic function and reactive oxygen intermediate production. SLP-76(-/-) and SLP-76(+/+) BMM secrete comparable levels of IL-12 in response to lipopolysaccharide and IFN-gamma. To examine macrophage function in vivo, SLP-76(-/-) mice were challenged i.v. with Listeria monocytogenes. SLP-76(-/-) mice survive and efficiently contain the acute phase of infection similar to wild-type mice but exhibit a stable chronic infection attributed to the lack of mature T cells. These data show that, although SLP-76 is required to couple Syk family PTK activity to downstream mediators and effector functions in Fc gamma R-induced pathways in some cell types, activation of Fc gamma R-dependent pathways occurs independently of SLP-76 in BM     

FcRL3基因单核苷酸多态性与中国汉族强直性脊柱炎相关性研究

目的 探讨Fc受体样基因3(FcRL3)部分单核苷酸多态性(SNP)在中国汉族强直性脊柱炎(AS)发病中的作用.方法 选取AS患者160例,健康对照组170例.用Mass-ArrayTM SNP检测仪对FcRL3基因上的SNP位点:FcRL3-1(rs0158440)、FcRL3-2(rs2225828)、FcRL3-3(rs7528684)、FcRL3-4(rs11284799)、FcRL3-5(rs945635)、FcRL3-6(rs3761959)、FcRL3-7(rs2210913)、FcRL3-8(rs2282284)、FcRL3-9(rs2282283)SNP进行检测.单倍型构建采用PHASEv2.1软件完成.结果 在AS组中FcRL3-1-1651C、FcRL3-3-169C、FcRL3-6 Intron3A、FcRL3-7 Intron3A等位基因频率病例组高于对照组;FcRL3-2-641C、FcRI3-4-110A、FcRL3-9 Intron15A等位基因频率病例组低于对照组;FcRL3-6、FcRL3-5和FcRL3-8SNP等位基因两组比较差异无统计学意义.AS组中单倍型FcRL3-1→9 CTCGCAAAA、CCCGGGGAA、CCCGCAAAC、CTCGCAAAC频率明显高于对照组;FcRL3-1→9 CCTGGGGAA和TCCACAAGA频率明显低于对照组.结论 FcRL3基因与中国汉族AS的易感性相关.     

Size-dependent effect of IgA on the IgA Fc receptor (CD89)

The IgA Fc receptor (FcR; CD89) is expressed on several types of cells of the myeloid cell lineage. We investigated whether different sizes of heat-aggregated IgA (aIgA) bind to CD89 and subsequently induce cellular activation. As a model we used the murine B cell line IIA1.6 transfected with CD89 or IIA1.6 cells transfected with CD89 as well as with the FcR γ chain to study the binding of IgA to CD89. When these cells expressing CD89 were incubated with monomeric IgA, no significant binding of IgA to the cells was detectable by fluorescence-activated cell sorter analysis; however, incubation of the cells with aggregated IgA resulted in 93 ± 2% positive cells. Incubation of the cells with different sizes of IgA-containing aggregates revealed optimal binding with aggregates containing five to six molecules of IgA per aggregate. No difference was observed between the binding to CD89 of both IgA1- or IgA2-containing aggregates. Furthermore, the binding of aIgA was found to be CD89-specific, since the binding of IgA was completely inhibited by the CD89-specific monoclonal antibody My43 and no detectable binding occurred to the IIA1.6 parent cell line. Activation studies using interleukin-2 (IL-2) production as a marker, showed that the FcR γ chain is necessary to induce cellular activation. Only cells transfected with both CD89 and the FcR γ chain (CD89⁺/γ⁺) enhance the IL-2 production 10–12-fold upon stimulation with aggregates of IgA. Furthermore, triggering of CD89 only results in increase of intracellular calcium concentration ([Ca²⁺]_i) in cells co-expressing FcR γ chain. Mutation of the tyrosine residues in the FcR γ chain immunoreceptor tyrosine-based activation motif of the FcR γ chain abolishes this increase in [Ca²⁺]_i, indicating association and involvement of the FcR γ chain in CD89-mediated signaling.     

Effect of Fc receptor blocking on human natural and lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

The effect of Fc receptor (FcR) blocking by aggregated human gamma-globulin (AGG) was studied on natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. Cytotoxicity was measured by detachment from the monolayer of [³H]TdR-prelabelled HEp-2 cells. LDCC was evaluated in a 24 h assay at 50:1 effector-target cell ratio in the presence of 25 μg/ml concanavalin A (Con A). Under these conditions but without Con A considerable NCMC was not elicited by normal lymphocytes. FcR blocking by AGG treatment of effector cells resulted in a significant NCMC activity to HEp-2 targets. In contrast, AGG treatment profoundly depressed LDCC. Monocyte depletion of effector cells had no major influence on the effect of AGG on NCMC and LDCC activities. An interference of FcR blocking by AGG and LDCC in response to Con A is suggested.     

T lymphocyte development in the absence of Fcϵ receptor Iγ subunit: analysis of thymic-dependent and independent αβ and γδ pathways

During fetal development, early thymocyte progenitors transiently express low affinity Fc receptors for IgG (FcγR) of both FcγRII and III isoforms. Only the FcγRIII isoform requires association of an FcγRIII (CD16) α subunit with an FcϵRIγ homodimer for surface expression. To address the role of FcγR in ontogeny, we studied thymic development in FcϵRIγ^−/− mice. We find that day 14.5 CD4⁻CD8⁻ double-negative (DN) fetal thymocytes of FcϵRIγ^−/− mice express mRNA of both FcγRIIb₁ and FcγRIII. Surface expression of FcγRII/III is readily detected on these cells. It appears that FcγRIIb₁, whose surface expression is FcϵRIγ independent, replaces FcγRIII during thymic development in these animals. Moreover, subsequent development into CD4⁺CD8⁺ double-positive and CD4⁺CD8⁻ and CD4⁻CD8⁺ single-positive subsets appears normal even in the absence of FcϵRIγ. However, alterations were noted in adult animals among the DN αβ TCR⁺ thymocytes and peripheral splenic DN T cells as well as CD8αα⁺ intestinal intraepithelial lymphocytes (iIEL). In contrast to conventional T lymphocytes, which do not express either FcγRIII or FcϵRIγ, DN αβ TCR⁺ thymocytes and extrathymically derived αβ TCR⁺ and γδ TCR⁺ CD8αα⁺β⁻ iIEL express TCR which incorporate FcϵRIγ as one of their subunits. Consistent with this, the TCR levels of these cells are lower than the TCR levels on cells from wild-type C57BL/6 mice. Despite the reduction in the level of surface TCR, the development of these cells was unaltered by the absence of FcϵRIγ. Thus, we observed alterations in adult DN αβ TCR⁺ thymocytes, splenic DN αβ TCR⁺ and DN γδ TCR⁺ large granular lymphocytes (LGL), and αβ TCR⁺ and γδ TCR⁺ CD8αα⁺β⁻ iIEL, but no detectable changes in their major fetal thymic developmental pathways. Cultivation of peripheral DN αβ TCR⁺ and DN γδ TCR⁺ cells from FcϵRIγ^−/− mice with interleukin-2 generates LGL which mediate natural killer activity. Unlike LGL from wild-type C57BL/6 mice, LGL from FcϵRIγ^−/− mice lack FcγRIII expression and could not mediate antibody-dependent cellular cytotoxicity through FcγRIII.     

Vessel-associated myogenic precursors control macrophage activation and clearance of apoptotic cells

Swift and regulated clearance of apoptotic cells prevents the accumulation of cell remnants in injured tissues and contributes to the shift of macrophages towards alternatively activated reparatory cells that sustain wound healing. Environmental signals, most of which are unknown, in turn control the efficiency of the clearance of apoptotic cells and as such determine whether tissues eventually heal. In this study we show that vessel-associated stem cells (mesoangioblasts) specifically modulate the expression of genes involved in the clearance of apoptotic cells and in macrophage alternative activation, including those of scavenger receptors and of molecules that bridge dying cells and phagocytes. Mesoangioblasts, but not immortalized myoblasts or neural precursor cells, enhance CD163 membrane expression in vitro as assessed by flow cytometry, indicating that the effect is specific. Mesoangioblasts transplanted in acutely or chronically injured skeletal muscles determine the expansion of the population of CD163⁺ infiltrating macrophages and increase the extent of CD163 expression. Conversely, macrophages challenged with mesoangioblasts engulf significantly better apoptotic cells in vitro. Collectively, the data reveal a feed-forward loop between macrophages and vessel-associated stem cells, which has implications for the skeletal muscle homeostatic response to sterile injury and for diseases in which homeostasis is jeopardized, including muscle dystrophies and inflammatory myopathies.     

On the interaction between agalactosyl IgG and Fcγ receptors

One of the serum abnormalities observed in autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) is the occurrence of IgG that lacks the terminal galactose on asparagine-linked biantennary complex type oligosaccharides [Gal(0)-IgG] located in the CH2 domain. Additionally, IgG without glycosylation is known to be defective in several effector functions due to a reduced ability to bind to its specific receptors (FcγR). It has thus been speculated that, by analogy with unglycosylated IgG, Gal(0)-IgG may also be functionally impaired or exert altered effector mechanisms. If this were true, Gal(0)-IgG could contribute to the phenotype of above-mentioned autoimmune diseases, like impaired immune complex clearance and defective down-regulation of activated B cells. Here, we show by three different methods that the interaction of Gal(0)-IgG and normally glycosylated IgG with the low-affinity FcγRII (CD32) is indistinguishable with respect both to binding and receptor-mediated signalling. These data argue against a prominent role for FcγR-dependent Gal(0)-IgG interactions in the etiology or pathogenesis of autoimmune diseases.     

Targeted IgA Fc receptor I (FcαRI) therapy in the early intervention and treatment of pristane‐induced lupus nephritis in mice

The Fc receptor I for IgA (FcαRI) down‐regulates humoral immune responses and modulates the risk of autoimmunity. This study aimed to investigate whether FcαRI targeting can affect progression of pristine‐induced lupus nephritis. In the first experiment (early intervention), four groups of animals were evaluated: untreated FcαRI/FcRγ transgenic (Tg) mice and Tg mice administered control antibody (Ctr Fab), saline and anti‐FcαRI Fab [macrophage inflammatory protein (MIP)‐8a], respectively, three times a week for 29 weeks, after being injected once intraperitoneally with 0·5 ml pristane. In the second experiment, antibody injection started after the onset of nephritis and was carried out for 2 months, with similar groups as described above. MIP‐8a improved proteinuria, decreased the amounts of glomerular injury markers, serum interleukin (IL)‐6, IL‐1 and monocyte chemoattractant protein (MCP)‐1, and F4/80 macrophages in the interstitium and glomeruli, in both experiments. When MIP‐8a was used as early intervention, a decrease in mouse serum anti‐nuclear antibody (ANA) titres and reduced deposition of immunoglobulins in glomeruli were observed. This effect was associated with reduced serum titres of immunoglobulin (Ig)G2a but not IgG1, IgG2b and IgG3. Furthermore, pathological analysis showed lower glomerular activity index and less fibronectin in MIP‐8a treated mice. This study suggests that FcαRI targeting could halt disease progression and lupus activation by selective inhibition of cytokine production, leucocyte recruitment and renal inflammation. Our findings provide a basis for the use of FcαRI as a molecular target for the treatment of lupus.