Electrokinetic study of the reactions of peritoneal macrophages and eosinophils with IgG immune complexes.

Electrophoretic light scattering (laser Doppler electrophoresis) has been employed to study the effects of guinea pig IgG immune complexes on the electrophoretic mobility distributions of guinea pig resident peritoneal cells. The resident population of cells is composed of macrophages (approximately 75%) and eosinophils (approximately 25%). These cells were separated according to the well-established method of Boyum. Populations of resident macrophages, eosinophils, and the unfractionated samples were incubated with soluble immune complexes, antigen alone, or antibody alone. The mean mobility of the resident macrophages decreased approximately 60% when incubated in the presence of immune complexes, although no effect could be discerned in the presence of antigen or antibody alone. The width of the resulting macrophage mobility distribution was larger than that of the control distributions, with a broad shoulder on the high-mobility side, indicating a heterogeneous response of the macrophages to the immune complexes. Eosinophils react in two distinct fashions. One population of eosinophils is present near the control experiments. The second population reacts in a manner very similar to that of macrophages. This suggests that at least two populations of eosinophils are present in the unstimulated guinea pig peritoneal cavity. Results that are intermediate between these two cases are found when unfractionated samples are studied.     

Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.     

Bombesin-like immunoreactivity in eosinophils. A light and electron microscopic study: effect of fixatives and cross-reactivity with various compounds.

Eosinophils immunopositive for bombesin tetradecapeptide were detected by means of light and electron microscopy in human and rat gastrointestinal tract and pulmonary tissue. This immunoreaction was only evidenced after the use of acetic acid-containing fixative such as Bouin's fluid. The dependence of this immunostaining on fixatives and time course were extensively studied. This immunoreaction promotes mainly one epitope probably associated with the C-terminal sequence. This epitope seems also to be present in other neuropeptides such as substance P (SP) and, to a lesser extent in chemotactic factors like formyl peptide (fMLP) or eosinophil chemotactic factor of anaphylaxis (ECF-A). At the electron microscopic level, the immunopositivity was associated with eosinophil membranes.     

Phorbol myristate acetate induces the secretion of an elastase by populations of resident and elicited mouse peritoneal macrophages

Cultured thioglycollate-elicited mouse peritoneal macrophages secrete an enzyme which hydrolyzes [³H]elastin prepared by NaB³H₄ reduction of bovine ligamentum nuchae elastin. Over a 24 h culture period, 3.5 · 10⁶ thioglycollate-elicited cells secrete sufficient enzyme to solubilize 200–350 μg [³H]elastin in 18 h. Secretion at this rate continues for at least 6 days in culture. Secretion of the enzyme is stimulated 3-fold by exposure of the cultured cells to 10^?7 M phorbol myristate acetate, whereas the parent alcohol 4α-phorbol is inactive in this respect. Enzyme activity is linearly related to the amount of conditioned medium assayed and is linear over incubation times up to 30 h. Unlabelled elastin competitively inhibits the solubilization of [³H]elastin. The solubilization rate is doubled if the substrate is pretreated with sodium dodecyl sulfate, but the rate of solubilization of this pretreated substrate increases with time. Resident peritoneal macrophages secrete barely detectable amounts of elastase, but phorbol myristate acetate (10^?7 M) stimulates its secretion in amounts comparable to those secreted by phorbol myristate acetate-stimulated thioglycollate-elicited cells. Dexamethasone (10^?9 M) inhibits phorbol myristate acetate-induced secretion by 50%, but 10^?6 M indomethacin is without effect. The secreted enzyme has the characteristics of a metalloproteinase.     

Electrokinetic response of granulocytes to concanavalin A and succinyl-concanavalin A

Electrophoretic light scattering has been used to study the effects of concavalin A (Con A) and succinyl-Con A on the electrophoretic mobility distribution of resident guinea-pig peritoneal eosinophils and human peripheral blood polymorphonuclear leukocytes. In both cell types, incubation with Con A (a tetrameric lectin) decreases slightly the mean mobility and increases substantially the width of the electrophoretic mobility distribution. These effects can be abolished by α-methyl-D-mannoside, a hapten sugar of Con A. Succinyl Con A, a dimeric derivative, was found to have no effect on the mobility distribution. These results are strikingly similar to our previous report of the response of the resident guinea-pig macrophage (19), suggesting possible parallels in the endocytic mechanisms of these cell types.     

Deletion of the miR-25/93/106b cluster induces glomerular deposition of immune complexes and renal fibrosis in mice

IgA nephropathy (IgAN), the most common form of primary glomerulonephritis, is caused by immune system dysfunction and affects only the kidneys. miRNA was involved in IgAN, in which their roles are still unknown. Herein, we found increased glomerular medulla size, proteinuria, kidney artery resistance, kidney fibrosis and immune complex deposition in 5-month miR-25/93/106b cluster knockout (miR-TKO) mice. In vitro, the inhibition of miR-25 cluster could promote cell proliferation and increase fibrosis-related protein and transferrin receptor (TFRC) expression in human renal glomerular mesangial cell (HRMC). Luciferase assay revealed that inhibition of miR-93/106b cluster could upregulate Ccnd1 expression through direct binding with the 3’UTR of Ccnd1. Conversely, inhibition of Ccnd1 expression prevented miR-93/106b-induced effect in HRMC. These findings suggested that miR-25 cluster played an important role in the progression of IgAN, which provided new insights into the pathogenesis and treatment of IgAN.     

Assessment of three simple techniques for the screening of circulating immune complexes: Correlation with a parameter of complement consumption

The sera of 36 normal controls, 45 patients with various diseases and 11 pregnant women were screened for circulating immune complexes using three relatively simple and inexpensive techniques. These included inhibition of agglutination of IgG coated latex particles with a serum having rheumatoid factor activity, polyethylene glycol precipitation and anti-complementary activity test. The circulating immune complexes were detected in a significantly higher proportion of patients as compared to normal controls. In the patients, the presence of circulating immune complexes did not always correlate with clinically detectable immunoinflammatory tissue damage indicating that pathogenic as well as nonpathogenic immune complexes were being detected by the above mentioned techniques. The alpha-1-antitrypsin/C3 ratio, however, correlated well with clinically apparent immuno-inflammation.     

The connective connection: interstitial cells of Cajal (ICC) and ICC-like cells establish synapses with immunoreactive cells. Electron microscope study in situ

We present transmission electron microscope (TEM) evidence that ICC and ICC-like cells frequently establish close contacts (synapses) with several types of immunoreactive cells (IRC): lymphocytes, plasma cells, eosinophils, basophils, macrophages and mast cells. Such synapses were found in various organs: human mammary gland and myometrium, as well as rat stomach, gut, bladder and uterus. Specimens were observed by conventional TEM on ultrathin sections. Based on morphometric analyses and computer-aided 3-D reconstructions from serial sections, we propose an operational definition of ICC-IRC synapses: cell-to-cell close contacts where the two cells are separated by only approximately 15 nm, equivalent to twice the plasmalemmal thickness. Two types of such synapses were found: (i) uniform ('plain') synapses (PS). close contact extending for >200 nm, and (ii) multi-contact ('kiss and run') synapses (MS)--with multiple, focal, close-contact points alternating with regions of wider intermembrane distance. For instance, a typical PS between a rat bladder ICC-like cell and an eosinophil was 2.48 microm long and 11+/-4 nm wide. By contrast, a MS synapse in rat myometrium (between an ICC-like cell and an eosinophil) was 8.64 microm long and had 13 contact points. The synaptic cleft measured 15+/-8 nm at contact points and approximately 100 nm or more in wider areas. These synapses are different from gap junctions usually seen between ICC and between ICC and smooth muscle cells. We previously proposed that ICC-like cells might represent stromal progenitor cells, participate in juxtacrine/paracrine signaling and play a role in immune surveillance. The nanoscopic distances between the two contiguous membranes suggest a juxtacrine cell-to-cell signaling (chemical synapse), via juxtacrinins, a specific case of phenomenins. However, the (micro)vesicles found in the synaptic cleft may correspond to an exosome-based mechanism.     

Comparative EPR study on high-spin ferric porphine complexes and cytochrome P-450 having rhombic character

Comparative EPR studies were made on two high-spin Fe(III) porphine model systems and mammalian liver microsomal cytochromes P-450, all of which exhibit approximately the same degrees of rhombicity in their EPR spectra. Comparison of g values and linewidths as a function of temperature, and of the microwave power saturation demonstrated that EPR characteristics of P-450 are more similar to the Fe(III) porphines having the thiolate axial ligand than in the other model systems, the mixed crystals of Fe(III) porphine with the corresponding free base porphine, in which no thiolate ligand is involved.There is, however, a discrepancy between P-450 and the model thiolates with respect to the size of the zero-field parameter D. These observations indicate that P-450 heme has essential structural features in common with thiolates but the Fe-S bond of P-450 may be modified from its normal orientation in model thiolates, probably as a result of the constraints imposed by the protein stucture.     

On the control between cell-mediated, IgM and IgG immunity

An hypothesis is proposed here describing some of the conditions that determine the type of response an antigen will induce, and explaining how the induction of one type of immunity affects the induction of other types of immunity. In more detail, the hypothesis attempts to account for the following observations: Some antigens induce only cell-mediated immunity, whereas others can, under different conditions, induce either cell-mediated or humoral immunity. The humoral response to most antigens consists of an initial period of IgM antibody synthesis, followed by a period of IgG synthesis. Some polymeric antigens induce the synthesis of only IgM antibody. There is a tendency for the immune response to an antigen, at a particular time, to be exclusively of the cell-mediated, IgM or IgG type.The hypothesis may also be relevant to some observations that, I believe, have been incorrectly interpreted to mean that “tolerance” to some antigens requires the presence of T (thymus-derived) cells specific for these antigens. The hypothesis suggests teleological reasons for the existence of the different types of immunity. It also suggests ways of controlling the type of response an antigen induces.     

The role of cystatins in cells of the immune system

The cystatins constitute a large group of evolutionary related proteins with diverse biological activities. Initially, they were characterized as inhibitors of lysosomal cysteine proteases - cathepsins. Cathepsins are involved in processing and presentation of antigens, as well as several pathological conditions such as inflammation and cancer. Recently, alternative functions of cystatins have been proposed: they also induce tumour necrosis factor and interleukin 10 synthesis and stimulate nitric oxide production. The aim of the present review was the analysis of data on cystatins from NCBI GEO database and the literature, and obtained in microarray and serial analysis of gene expression (SAGE) experiments. The expression of cystatins A, B, C, and F in macrophages, dendritic cells and natural killer cells of the immune system, during differentiation and activation is discussed.     

The PsbS protein controls the macro-organisation of photosystem II complexes in the grana membranes of higher plant chloroplasts

The PsbS protein is a critical component in the regulation of non-photochemical quenching (NPQ) in higher plant photosynthesis. Electron microscopy and image analysis of grana membrane fragments from wild type and mutant Arabidopsis plants showed that the semi-crystalline domains of photosystem II supercomplexes were identical in the presence and absence of PsbS. However, the frequency of the domains containing crystalline arrays was increased in the absence of PsbS. Conversely, there was a complete absence of such arrays in the membranes of plants containing elevated amounts of this protein. It is proposed that PsbS controls the macro-organisation of the grana membrane, providing an explanation of its role in NPQ.     

A polyamine- and LHCII protease activity-based mechanism regulates the plasticity and adaptation status of the photosynthetic apparatus

In the present study we aim to dissect the basis of the polyamine mode of action in the structure and function of the photosynthetic apparatus. Although the modulating effects of polyamines in photosynthesis have been reported since long [K. Kotzabasis, A role for chloroplast-associated polyamines? Bot. Acta 109 (1996) 5-7], the underlying mechanisms remained until today largely unknown. The diamine putrescine was employed in this study, by being externally added to Scenedesmus obliquus cultures acclimated to either low or high light conditions. The results revealed the high efficiency by which putrescine can alter the levels of the major photosynthetic complexes in a concerted manner inducing an overall structure and function of the photosynthetic apparatus similar to that under higher light conditions. The revealed mechanism for this phenomenon involves alterations in the level of the polyamines putrescine and spermine which are bound to the photosynthetic complexes, mainly to the LHCII oligomeric and monomeric forms. In vitro studies point out to a direct impact of the polyamines on the autoproteolytic degradation of LHCII. Concomitantly to the reduction of the LHCII size, exogenously supplied putrescine, induces the reaction centers' density and thus the photosynthetic apparatus is adjusted as if it was adapted to higher light conditions. Thus polyamines, through LHCII, play a crucial role in the regulation of the photosynthetic apparatus' photoadaptation. The protective role of polyamines on the photosynthetic apparatus under various environmental stresses is also discussed in correlation to this phenomenon.     

IgGs containing light chains of the λ and κ type and of all subclasses (IgG1‐IgG4) from sera of patients with multiple sclerosis hydrolyze DNA

We present the first evidence demonstrating that small fractions of IgGs of all four subclasses (IgG1–IgG4) are catalytically active in the hydrolysis of DNA and on average their relative activity (nM supercoiled DNA/1mg IgG/1 h) increases in the order: IgG1 (0.58) < IgG2 (0.94) < IgG3 (1.4) < IgG4 (4.1), while their approximate relative contribution to the total activity of abzymes increases in the order: IgG1 (6.9%) < IgG3 (9.3%) < IgG2 (18.2%) < IgG4 (65.6%). On average IgGs containing light chains of the λ‐type are severalfold more active in the hydrolysis of DNA than IgGs with light chains of the κ‐type. Using different physicochemical methods of antibody analysis we have shown that the immune system of multiple sclerosis patients generates a variety of anti‐DNA abzymes of different type and with different catalytic properties, which can play an important role in multiple sclerosis pathogenesis. Copyright © 2010 John Wiley & Sons, Ltd.     

Heterogenous populations of cytotoxic cells in the peritoneal cavity of BALB/c mice immunized with allogeneic EL4 leukemia cells

Adherent cells, presumably macrophages, obtained from the peritoneal cavity shortly after rejection of the allogeneic leukemia EL4, produced effective cell-mediated cytotoxicity (CMC) in vitro. These cytotoxic cells were sensitive to anti-macrophage serum and resistant to anti-thymocyte serum and 10,000 roentgen irradiation. In contrast, a second population of specifically cytotoxic cells were nonadherent, sensitive to x-rays and anti-thymocyte serum, but not to anti-macrophage serum.The two cell populations had a cooperative cytotoxic effect in vitro against allogeneic tumor cells.     

IgG and IgE immune response against the surface glycoprotein gp200 of Saccharomyces cerevisiae in patients with atopic dermatitis

The heat-stable and soluble glycoprotein gp200 (molecular weight 200 kDa) is part of the cell wall of S. cerevisiae. Recently, an association was shown between IgA and IgG against gp200 and inflammation in Crohn's disease. Gp200 is able to induce a proliferation of human lymphocytes in vitro, together with a natural killer cell associated cytotoxicity. Specific IgE against Saccharomyces cerevisiae (baker's or brewer's yeast) may be detected in approximately 73 %, against Candida albicans in 68% of those patients suffering from severe atopic dermatitis. The aim of this study was to elucidate the possible role of an anti-gp200 immune response for the pathogenesis of atopic dermatitis by immunoblot analysis. Anti-gp200 IgE was found in 55% of healthy individuals, in 67% of individuals with atopic predisposition without eczema, in 63% of the patients with mild atopic dermatitis, and in 86% of patients with severe atopic dermatitis, respectively. On the contrary, anti-gp200 IgG could be shown in 55% of healthy individuals, in 89% of individuals with atopic predisposition but without eczema, in 100% of patients with mild atopic dermatitis, and in 79% with severe atopic dermatitis, respectively. No immunoreactivity was found when an extract of Arxula adeninivorans was used as antigen. These results underline the specificity of the immunoblot results with gp200 from Saccharomyces cerevisiae. It can be concluded that occurrence of specific IgE against Saccharomyces cerevisiae cannot be explained by a cross reactivity, e.g., against Candida albicansallergens. Further investigations with the recombinant gp200 will give information on the role of this glycoprotein both in atopic dermatitis and Morbus Crohn. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chip-based gel electrophoresis method for the quantification of half-antibody species in IgG4 and their by- and degradation products

The inter-heavy-chain disulfide bonds of the IgG4 subclass can be described as being at equilibrium with the intra-chain disulfide bonds. This means that a fraction of IgG4 has noncovalently linked heavy chains (half-antibody). The percentage of half-antibodies produced depends upon the expression system used. Nondenaturing assays fail to separate the half-antibodies from the native form because two half-molecules are held together by noncovalent forces. The pharmaceutical industry needs a reliable denaturing assay for checking batch-to-batch consistency. Until now sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been the standard method used to detect the presence of half-antibodies. However, this technique is laborious and cannot be automated. Furthermore, cumbersome densitometric measurements are necessary for quantification. To overcome these disadvantages a chip-based gel electrophoresis method was investigated. In the nonreduced format the separation profile is compared with that from SDS-PAGE. The limit of quantification as a percentage of the amount applied, repeatability, reproducibility, and linearity are compared with those of SDS-PAGE. The amounts of half-antibody and of by- and degradation products were determined for several batches by using area percentage and by external calibration with IgG4 as a reference standard. Both methods allow avoidance of error introduction for the quantification as is the case by application of myosin as reference concentration. Both sets of results are compared with each other and with the results from SDS-PAGE. In the reduced format it is noted that the reduction of the inter-heavy-chain disulfide bridges proceeds faster than the reduction of the heavy-light-chain bonds. Therefore optimized conditions are necessary to obtain a complete reduction.     

Asia I型口蹄疫病毒中和表位与羊IgG重链恒定区的融合表达及免疫原性测定

VP1蛋白是口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)诱导机体产生抗病毒感染免疫的主要蛋白,含有病毒的若干中和表位。本研究设计和合成了由AsiaI型FMDVVP1蛋白136~160aa和198~211aa两个表位组成的重复串联表位的编码基因,并克隆了羊IgG重链恒定区编码基因。利用BamHI、EcoRI和XhoI位点将2个基因片段依次克隆到pPROExHTb载体,构建成重组质粒pPRO-FshIgG,将其转化大肠杆菌BL21(DE3)感受态细胞,以IPTG诱导表达得到融合蛋白FshIgG。100μgFshIgG蛋白免疫豚鼠后刺激豚鼠产生了高效价的FMDV中和抗体,而且使这些免疫豚鼠在用200ID50剂量FMDV攻击时得到了完全保护。由此证明,羊IgG重链恒定区蛋白能够作为FMDV表位肽的载体,而融合蛋白FshIgG可成为一种口蹄疫表位疫苗候选物用于口蹄疫的预防。