Possible role of factor XIII subunit A in Fcgamma and complement receptor-mediated phagocytosis

Besides its traditional role in hemostasis, factor XIII subunit A (FXIII-A) is supposed to function as a cellular transglutaminase and to be involved in certain intracellular processes, including cytoskeletal remodeling. To investigate its intracellular role, the aim of the present study was to follow changes in FXIII-A production in combination with the receptor-mediated phagocytic activities of monocytes/macrophages and to examine the phagocytic functions of monocytes in patients with FXIII-A deficiency. Human blood monocytes were isolated from the buffy coats of healthy volunteers and cultured for 4 days. The FcgammaR-mediated phagocytosis of sensitized erythrocytes (EA) and the complement receptor (CR)-mediated phagocytosis of complement-coated yeast particles were studied during monocyte/macrophage differentiation. Changes in the gene expression of FXIII-A were detected by real-time quantitative RT-PCR. FXIII-A protein production was investigated with fluorescent image analysis at single cell level and Western immunoblot analysis. Both the FcgammaR and CR-mediated phagocytosis increased during culturing, which peaked on day 3. The phagocytic activity of the cells could be markedly inhibited with monodansylcadaverine, an inhibitor of the transglutaminase-induced crosslinking of proteins. The phagocytosis of EA, complement-coated and uncoated yeast particles was found to be strongly diminished in monocytes of FXIII-A deficient patients. The phagocytic functions of cultured cells showed a change in parallel with the alterations in FXIII-A mRNA expression, as well as with that in FXIII-A in protein synthesis detected by image and Western immunoblot analyses in concert. Our results suggest that FXIII-A plays a role in the Fcgamma and complement receptor-mediated phagocytic activities of monocytes/macrophages.     

Expression of tumour necrosis factor receptors (CD120a and CD120b) on bronchoalveolar cells.

It is generally accepted that physiological modulators for tumour necrosis factor (TNF) are present in a variety of body fluids including serum. Among these modulators are soluble TNF receptors (TNF-R) that are cleaved from the extracellular domain of the TNF-Rs. Two receptors of different structures with molecular weights of 55 kDa (CD120a) and 75 kDa (CD120b) are known to be expressed on monocytes, lymphocytes, granulocytes and other cells of peripheral blood. The aim of our study was to determine the expression of CD120a and CD120b on bronchoalveolar lavage cells (BAL cells). BAL cells of 14 patients with different pulmonary disorders were stained with anti-CD120a and anti-CD120b monoclonal antibodies and were differentiated by FACS analysis. Both TNF-Rs are expressed on monocytes, macrophages, lymphocytes and granulocytes of the BAL. Although the relation of CD120a to CD120b is individual for a given cell type and an individual patient, strict correlations between both receptors were observed for BAL monocytes and alveolar macrophages. CD120a are expressed on 29.7% of alveolar macrophages; similar data were obtained for CD120b. 24.3% of the BAL monocytes were positive for CD120a and 25.5% for CD120b. 4.1% of the BAL lymphocytes were positive for CD120a whereas the percentage of CD120b positive BAL lymphocytes was approximately six times greater. Analysis of BAL granulocytes revealed 21.2% cells positive for CD120a and 11.6% for CD120b. In contrast to the BAL cells named above there was no positive correlation between CD120a and CD120b expression on BAL lymphocytes and granulocytes. We were able to show that TNF-Rs of BAL cells, like those of blood cells, are shedded in vitro after incubation with or without lipopolysaccharide (LPS), detected as TNFalpha-inhibitor activity in cell culture supernatant. In conclusion, BAL cells express and shed TNF-Rs, as is known for cells of other body compartments.     

Effect of the EM Bokashi® Multimicrobial Probiotic Preparation on the Non-specific Immune Response in Pigs

The aim of the study was to determine the effect of EM Bokashi® on the phagocytic activity of monocytes and granulocytes, oxidative burst, SWC3, and CD11b + CD18+ expression on monocytes and granulocytes, and the serum concentration of cytokine and lysozyme in pig. 60 Sixty female piglets were divided into two groups: I – control and II – experimental. For the experimental group, a probiotic in the form of the preparation EM Bokashi® was added to the basal feed. Flow cytometry was used to determine selected non-specific immune response parameters, intracellular production of hydrogen peroxide by peripheral granulocytes and monocytes, and surface particles in peripheral blood. The EM Bokashi® preparation used in the study was found to increase phagocytic activity mainly in monocytes, with an increased percentage of phagocytic cells in the experimental group. The highest serum lysozyme concentration in the piglets in the experimental group (2.89 mg/dl), was noted on day 42 of the study. In the group of pigs receiving EM Bokashi®, the percentage of phagocytic cells with SWC3 (monocyte/granulocyte) expression was statistically significantly higher than in the control. The increase in the number of cells with SWC3 (monocyte/granulocyte) expression in the peripheral circulation in combination with the greater capacity of the cells for phagocytosis and respiratory burst confirms that the non-specific immune response was modulated in the pigs supplemented with EM Bokashi®.

     

Phagocytic capacity of Kupffer cells during hepatic amoebiasis in guinea pigs.

Very few studies have been carried out on the role of liver macrophages (Kupffer cells) during the course of hepatic amoebiasis. The kinetics of phagocytic activity of Kupffer cells and blood monocytes was studied in guinea pigs intra-mesenterically infested with Entamoeba histolytica. The phagocytic capacity of blood monocytes of normal animals was comparatively lower than Kupffer cells for both latex and haemolysin coated sheep red blood cells. Significant decline in phagocytic response of Kupffer cells and blood monocytes of infected animals was observed right from 2nd post infection day and it kept on decreasing with the progress of infection. Depression in phagocytic response of Kupffer cells and blood monocytes was more marked in those animals who had higher grades of pathological lesions. Hence, an inverse correlation was obtained between the phagocytic capacity and severity of amoebic lesions (P less than 0.01). The significance of depression in phagocytic response of Kupffer cells and blood monocytes may be responsible for the development of hepatic lesions.     

Origin and Kinetics of Resident Tissue Macrophages

Abstract To elucidate the origin and renewal kinetics of peritoneal macrophages, as a typical example of the mononuclear phagocytic system, syngeneic rats were treated with tritiated thymidine [³H]TdR and leucocytes were transferred to unlabelled recipients over a bilateral arteriovenous shunt. Labelled and unlabelled monocytes were evenly distributed in both animals as shown by autoradiography. It was ascertained that no ‘autoradiographically’ detectable reutilization of label occurred and that transferred cells showed undisturbed kinetics. the results imply: 1 resident peritoneal macrophages derive from blood monocytes; 2 peritoneal macrophages represent a homogeneous population in respect to their cellular origin; 3 blood monocytes as a myelogeneous cell line do not represent a generative end cell. They migrate into the tissue (peritoneal cavity) and differentiate into resident macrophages, undergoing on average one mitosis per cell during a period of approximately 7 days. 4 resident peritoneal macrophages are derived 50% from blood monocytes and 50% from division in situ; and 5 under steady-state conditions the renewal rate amounts to 0. 18%/h, which yields a half-life time of 16 days and a renewal time of 23 days.     

Neuropeptide Y modulates functions of inflammatory cells in the rat: distinct role for Y1, Y2 and Y5 receptors

Neuropeptide Y (NPY) has been reported to be a potent anti-inflammatory peptide with ability to directly modulate activity of granulocytes and macrophages. The present study aimed to correlate the effects of NPY in vivo on lipopolysaccharide-induced air-pouch exudates cells and in vitro on peripheral blood leukocytes functions. The role of different Y receptors was examined using NPY-related peptides and antagonists with diverse subtype specificity and selectivity for Y receptors. Y1, Y2 and Y5 receptors were detected on air-pouch exudates cells (flow cytometry) and peripheral blood granulocytes (immunocitochemistry). NPY in vivo reduced inflammatory cells accumulation into the air pouch, and decreased their adherence and phagocytic capacity via Y2/Y5 and Y1/Y2 receptors, respectively. Quite the opposite, NPY in vitro potentiated adhesiveness and phagocytosis of peripheral blood granulocytes and monocytes by activating Y1 receptor. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay.     

The effect of Bilirubin on the phagocytic activity of mouse peripheral granulocytes and monocytesin vivo

Using an in vitro phagocytic assay with synthetic 2-hydroxyethylmethacrylate copolymer particles, the phagocytic activity of leukocytes of bilirubin-treated mice (85 and 170 mumol/L in 0.5 mL intraperitoneally) was studied. Bilirubin treatment significantly stimulated the phagocytosis of both peripheral blood granulocytes and monocytes; the increase of phagocytosis persisted for 6 h after bilirubin injection. The potential immunostimulating and/or immunotoxic effect of bilirubin is discussed.     

Phagocytic function in cyclists: correlation with catecholamines and cortisol.

Flow cytometer measurements were made of the basal variations in peripheral blood functional monocytes and granulocytes over the course of a training season (January to November) of a cycling team. Parallel determinations were made of plasma concentration of catecholamines (chromatography) and cortisol (RIA) in a search for neuroendocrine markers. The results showed the greatest phagocytic capacity to occur in the central months (March, May, and July), coinciding with the greatest number and highest level of competitive events with good correlation with a peak in epinephrine during these months (r(2) = 0.998 for monocytes and r(2) = 0.674 for granulocytes). No good correlations were found between phagocytosis and norepinephrine or cortisol. The highest values for phagocytosis and epinephrine concentration were found in May. These results suggest that blood epinephrine concentration could be a good neuroendocrine marker of sportspeople's phagocytic response.     

Differentiation of granuloma cells (epithelioid cells and multinucleated giant cells): a morphometric analysis. Investigations using the model of experimental autoimmune (anti-TBM) tubulo-interstitial nephritis

Morphometric analysis disclosed distinct differences between blood monocytes, tissue monocytes (i.e. immature macrophages), epithelioid cells and multinucleated giant cells as well as phagocytic macrophages (i.e. mature macrophages) in the granuloma model of autoimmune (anti-TBM) tubulo-interstitial nephritis. The numerical density of lysosomes decreased slightly in tissue monocytes compared with blood monocytes but showed a pronounced increase during the formation of epithelioid cells. The lysosomal compartments of epithelioid cells and multinucleated giant cells resembled each other very closely, but the giant cells obviously produced additional lysosomes of small diameter (80-120 nm). Phagocytic macrophages displayed a total numerical density of lysosomes similar to that of tissue monocytes but the mean diameter of the lysosomes was markedly greater. Thus the volume density of lysosomes was highest in phagocytic macrophages. The blood monocytes exhibited the smallest lysosomal compartment. In tissue monocytes, epithelioid cells, and multinucleated giant cells the volume densities of the lysosomes were greater than in blood monocytes and remained relatively constant because the increase in numerical density was counterbalanced by a decrease in mean granule diameter. We found only minor differences in mitochondrial volume densities among the five cell populations. The shape of the mitochondria, however, changed steadily from short rotational ellipsoids in the blood monocytes to rather elongated and slender bodies in the multinucleated giant cells. The results suggest that epithelioid cells and multinucleated giant cells are active cells which may contribute by their specific performances, to the immunologic microenvironment of the granuloma.     

Unmodified phagocytosis after periodate treatment of human granulocytes separated by a two-step method using Ficoll-Hypaque and polyvinylic alcohol medium

A two-step method separating granulocytes and mononuclear cells from a single sample of 10 ml human blood is described. The technique combines a Ficoll-Hypaque treatment and a sedimentation in a Polyvinylic alcohol medium. This method (FHP) is then compared to the Ficoll-Hypaque-Dextran procedure. The FHP method requires approximately 2 h to be carried out and results in the preparation of granulocytes (greater than 99% purity) with high yield (40.7 % +/- 9.1). The cell viability exceeds 95%. Moreover the enzymic and phagocytic properties of polymorphonuclear cells are preserved after isolation. This method is then applied to study the influence of periodate treatment on nonspecific phagocytosis. In contrast with monocytes, we observed no decrease of phagocytic process.     

A monoclonal antibody recognising a surface marker on rainbow trout (Oncorhynchus mykiss) monocytes

The characterisation of a monoclonal antibody (mab 45) reacting with phagocytic leucocytes isolated from blood and spleen of rainbow trout (Oncorhynchus mykiss, Walbaum) is described. The surface marker labelled by this mab is expressed at relative low levels on the membrane of large, nearly nongranulated trout leucocytes, and having the typical morphology of monocytes in flow cytometry (Kfoury et al., 1999, Fish Pathology, 34, 1-6). No reaction of mab 45 with granulocytes, lymphocytes or thrombocytes was detected. In spleen and head kidney, large, polymorphonuclear leucocytes were immunostained. The mab most strongly recognised an antigen of 48 kDa prepared from trout leucocytes of different organs, but not in trout plasma. In an in vitro phagocytosis assay trout monocytes were stained with mab 45 after phagocytosis of Aeromonas salmonicida labelled with the lipophilic fluorescent cell surface linker PKH26. However, previous binding of mab 45 on trout leucocytes did not inhibit the phagocytosis of A. salmonicida particles. Using mab 45, the dynamics of monocytes in blood, spleen and peritoneal cavity could be demonstrated after intraperitoneal injection of trout with inactivated A. salmonicida. The described mab serves as a useful tool to investigate the involvement of monocytes/macrophages in immune reactions of trout to a variety of pathogens.     

Phagocytosis of bacterial magnetite by leucocytes

Summary Magnetotactic bacteria were introduced into granulocytes and monocytes by phagocytosis. The number of phagocytes containing bacterial magnetites (magneto-sensitive cells) became constant after 1.5 h incubation, and viable phagocytes contained about 20–40 cells of magnetotactic bacteria. Granulocytes and monocytes containing bacterial magnetites were separated by magnet a Samarium-cobalt from lymphocytes. After separation, 89% of lymphocytes were recovered and 95% of the cells were viable. The contamination of phagocytes in the recovered lymphocytes was below 0.8%. Magneto-sensitive granulocytes and monocytes were removed by applying a magnetic field. The nitro-blue tetrazolium-reducing, chemotactic and phagocytic abilities of phagocytes ingesting magnetotactic bacteria were 84%, 88% and 87% respectively after 1 h incubation.     

Antifungal activity of the local complement system in cerebral aspergillosis

Dissemination of aspergillosis into the central nervous system is associated with nearly 100% mortality. To study the reasons for the antifungal immune failure we analyzed the efficacy of cerebral complement to combat the fungus Aspergillus. Incubation of Aspergillus in non-inflammatory cerebrospinal fluid (CSF) revealed that complement levels were sufficient to obtain a deposition on the surface, but opsonization was much weaker than in serum. Consequently complement deposition from normal CSF on fungal surface stimulated a very low phagocytic activity of microglia, granulocytes, monocytes and macrophages compared to stimulation by conidia opsonized in serum. Similarly, opsonization of Aspergillus by CSF was not sufficient to induce an oxidative burst in infiltrating granulocytes, whereas conidia opsonized in serum induced a clear respiratory signal. Thus, granulocytes were capable of considerably reducing the viability of serum-opsonized Aspergillus conidia, but not of conidia opsonized in CSF. The limited efficacy of antifungal attack by cerebral complement can be partly compensated by enhanced synthesis, leading to elevated complement concentrations in CSF derived from a patient with cerebral aspergillosis. This inflammatory CSF was able to induce (i) a higher complement deposition on the Aspergillus surface than non-inflammatory CSF, (ii) an accumulation of complement activation products and (iii) an increase in phagocytic and killing activity of infiltrating granulocytes. However, levels and efficacy of the serum-derived complement were not reached. These data indicate that low local complement synthesis and activation may represent a central reason for the insufficient antifungal defense in the brain and the high mortality rate of cerebral aspergillosis.     

Occurrence of histone H1° protein in rat alveolar macrophages

Histone H1 of rat alveolar macrophages, neutrophilic granulocytes and monocytes extracted with 5% (v/v) perchloric acid was studied in order to see whether a protein similar to histone H1° of rat liver exists in these specialized cells. The biochemical methods used involved SDS and acid-urea polyacrylamide gel electrophoresis, gel filtration on BioGel P100 and raising antisera against chromatographically purified rat liver H1° and histone H1. The antiserum was applied for further characterization of the presumptive H1° fraction using ELISA and Western blot analysis.The results from our studies showed that histone H1° protein is present in rat alveolar macrophages, monocytes and neutrophilic granulocytes, but its quantity in neutrophilic granulocytes is very much less than macrophages and monocytes.     

流式检测PNH克隆的方法学探讨及临床筛检和意义

目的:探讨流式细胞仪高敏感方法检测PNH克隆的必要性和血细胞减少患者PNH克隆的发生率及临床意义。方法:采用CD59检测红细胞及FLAER联合CD24或CD14检测粒细胞和单核细胞的方法检测了20例健康志愿者和1 095例血细胞减少患者的PNH克隆比例,同时对31例患者采用传统的CD59方法检测粒细胞和单核细胞中CD59~-细胞比例。结果:根据健康志愿者正常背景和患者获取的细胞数,确定粒细胞、单核细胞和红细胞PNH克隆的最低检测限(LOD)分别为0.04%、0.10%和0.05%;827例患者FLAER~-CD24~-粒细胞、FLAER~-CD14~-单核细胞和CD59~-红细胞的中位比例分别为0.02%、0.02%和0.03%;318例(38.45%)患者粒细胞PNH克隆高于LOD,180例FLAER~-CD24~-粒细胞1.00%,粒红和粒单细胞的一致性只有43%～45%。138例FLAER~-CD24~-粒细胞≥1.00%,粒红、粒单和粒单红细胞的一致率分别为91.30%、97.83%和89.86%;比较CD59~-和FLAER~-CD24~-粒细胞、CD59~-和FLAER~-CD14~-单核细胞比例,CD59~-细胞比例明显更低(P0.000 1和P=0.000 9);26.85%和24.54%患者因出现FLAER~-CD24~+粒细胞与FLAER~-CD14~+单核细胞,导致FLAER单阴比例高于FLAER和锚连蛋白双阴比例,对PNH克隆比例低于0.10%的患者影响更大;36例PNH患者和50例MDS或AA患者PNH克隆比例分别为90.30%(44.49%～99.05%)和1.30%(0.10%～96.07%)(P0.000 1);PNH克隆比例等于41.81%时,诊断PNH的敏感度和特异度分别为100.0%和96.0%。结论:在本实验条件下根据正常背景值,确定粒细胞、红细胞的LOD分别为0.04%和0.05%。粒细胞PNH克隆比例≥1.00%,与单核和红细胞诊断PNH克隆阳性结果更一致,PNH克隆比例高于41.81%时PNH疾病可能性更大。     

Functional status of peripheral blood monocyte in patients with Ixodes tick-borne borreliosis accompanied by opisthorchiasis

The characteristics of the functional status of peripheral blood monocytes/macrophages in patients with Ixodes tick-borne acute borreliosis accompanied by opisthorchiasis invasion were studied. The study revealed a decrease in the phagocytic activity of monocytes and in the level of expression of cell receptors Fcgamma with the expression of cell receptors C3beta being normal.     

Glucan-like synthetic oligosaccharides: iterative synthesis of linear oligo-beta-(1,3)-glucans and immunostimulatory effects

Small reducing and linear oligo-beta-(1,3)-glucans, which are able to act as phytoallexin elicitors or as immunostimulating agents in anticancer therapy, were synthesized according to an iterative strategy that involved a unique key monosaccharidic donor. To avoid anomeric mixtures, the reducing entity of the target oligomers was first locked with benzyl alcohol and further selective deprotection of the 3-OH with DDQ afforded the desired building block as an acceptor. The latter was then used in a second cycle of glycosylation/deprotection to afford the desired disaccharide, and successive reiterations of this process provided the desired oligomers. Unusual conformational behaviors were observed by standard NMR sequences and supported by NOESY studies. Finally, removal of protecting groups afforded free tri-, tetra-, and pentaglucosides in good overall yields. Two oligosaccharides representing linear laminaritetraose and laminaripentaose were compared to the recently described beta-(1,3)-glucan phycarine. Following an intraperitoneal injection, the influx of monocytes and granulocytes into the blood and macrophages into the peritoneal cavity was comparable to that caused by phycarine. Similarly, both oligosaccharides stimulated phagocytic activity of granulocytes and macrophages. Using ELISA, we also demonstrated a significant stimulation of secretion of IL-1beta. Together these results suggest that the synthetic oligosaccharides have similar stimulatory effects as natural beta-(1,3)-glucans.     

Zebrafish Kidney Phagocytes Utilize Macropinocytosis and Ca2+-Dependent Endocytic Mechanisms

Background

The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including active endocytosis by macrophages and granulocytes. Endocytosis can be used as a reliable measure of selective and non-selective mechanisms of antigen uptake in the early phase of an immune response. Numerous assays have been developed to measure this response in a variety of mammalian and fish species. The small size of the zebrafish has prevented the large-scale collection of monocytes/macrophages and granulocytes for these endocytic assays.

Methodology/Principal Findings

Pooled zebrafish kidney hematopoietic tissues were used as a source of phagocytic cells for flow-cytometry based endocytic assays. FITC-Dextran, Lucifer Yellow and FITC-Edwardsiella ictaluri were used to evaluate selective and non-selective mechanisms of uptake in zebrafish phagocytes.

Conclusions/Significance

Zebrafish kidney phagocytes characterized as monocytes/macrophages, neutrophils and lymphocytes utilize macropinocytosis and Ca²⁺-dependant endocytosis mechanisms of antigen uptake. These cells do not appear to utilize a mannose receptor. Heat-killed Edwardsiella ictaluri induces cytoskeletal interactions for internalization in zebrafish kidney monocytes/macrophages and granulocytes. The proposed method is easy to implement and should prove especially useful in immunological, toxicological and epidemiological research.     

Ultrastructure of the peripheral blood leucocytes of Oreochromis mossambicus

Peripheral blood leucocytes were examined for their fine structure and their in vivo response to colloidal carbon particles and bacteria, Aeromonas salmonicida . Four main types of leucocyte were identified: lymphocytes, thrombocytes, monocytes and granulocytes. Previously, two types of granulocyte had been identified but at the electron microscope level a third type could be distinguished in the peripheral blood. Monocytes and the type 1 and type 3 granulocytes were found to be phagocytic.     

Flow Cytometric Characterization of Bovine Blood Neutrophil Phagocytosis of Fluorescent Bacteria and Zymosan Particles

A Flow Cytometric method for the evaluation of the phagocytic capacity of bovine blood neutrophils is described. The neutrophils were isolated from bovine blood by a one step discontinuous gradient of Percoll. By this technique of isolation, 90 ± 2.8 % (mean ± s) of the granulocytes in the whole blood were recovered. Isolated neutrophils were incubated with FITC labeled S. aureus or zymosan particles in a ratio of 1:20 and 1:10, respectively, and a final serum concentration of 10 %. Phagocytosis was terminated after 15 min and the number of extracellular bacteria or zymosan particles and the percentage of phagocytic granulocytes were registered by Flow Cytometry (FCM). FCM and microscopic studies revealed that eosinophils play a minor role in the phagocytosis of bacteria. The neutrophils were the main population of the granulocytes which were actively phagocytic. Variation among cows in the ability of their blood neutrophils to phagocytize bacteria was evident.