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。     

Analysis of PI (phosphatidylinositol)-anchoring antigens in a patient of paroxysmal nocturnal hemoglobinuria (PNH) reveals deficiency of 1F5 antigen (CD59), a new complement-regulatory factor.

FACS analysis together with PIPLC treatment was applied to PI-anchoring antigens such as DAF (decay-accerelating factor, CD55), 1F5 antigen (CD59), CD14 and CD16 on the cell surfaces of blood cells from a normal adult and a male patient with paroxysmal nocturnal hemoglubinuria (PNH). Through the extensive analysis, this patient proved to be completely defective in 1F5 antigen, a newly found complement-regulatory protein, on all the blood cells tested. In normal blood cells such as lymphocytes, monocytes and granulocytes, 1F5 antigen was expressed as one of PI-anchoring proteins. In contrast to most of PNH patients, this patient reserved DAF, CD14 and CD16 at normal levels in his erythrocytes, monocytes and granulocytes. Also, there were no significant differences between the normal adult and the patient in the activities of erythrocyte acetylcholinesterase and granulocyte alkaline phosphatase which were also known to be PI-anchoring enzymes. Thus, deficiency of 1F5 antigen must be deeply related to the clinical symptoms of PNH in this patient.     

Flow cytometric analysis of CD55 and CD59 expression on blood cells in paroxysmal nocturnal haemoglobinuria

PNH is a rare clonal disorder of hematopoietic stem cells, therefore all blood cells lineages are involved. The main feature is an increased sensitivity of erythrocytes to complement-mediated cell lysis due to deficiency of membrane-bound GPI (glycosylphosphatidylinositol)-anchored proteins which normally function as inhibitors of reactive hemolysis. In the present study, we performed flow cytometric analysis using monoclonal antibodies against CD55 and CD59 for the detection of PNH-type clone in the blood of 50 patients (28 females and 22 males, age range 7-67 yrs). In one patient only we found a large population (95%) of granulocytes with decreased expression of both CD55 and CD59 molecules (type I PNH) and in two others with partial loss of CD55 expression (type II PNH). The expression was determined chiefly on granulocytes which in the control group showed reliable and high expression of CD55 and CD59.     

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.     

Aurin Tricarboxylic Acid Protects against Red Blood Cell Hemolysis in Patients with Paroxysmal Nocturnal Hemoglobinemia

Objectives

Paroxysmal nocturnal hemoglobinemia (PNH) is a rare but serious condition characterized by complement-mediated red blood cell (RBC) hemolysis and episodic thrombotic attack. It results from decay accelerating factor (CD55), and protectin (CD59), becoming attached to RBC and other cell surfaces. Absence of these protective proteins leaves such cells vulnerable to self attack at the C3 convertase and membrane attack complex (MAC) stages of complement activation. We have previously reported that aurin tricarboxylic acid (ATA) is an orally effective agent that selectively blocks complement activation at the C3 convertase stage as well as MAC formation at the C9 insertion stage.

Design and Methods

We used a CH50 assay method and western blot analysis to investigate the vulnerability to complement attack of PNH RBCs compared with normal RBCs. Zymosan was used as the activator of normal serum and PNH serum. ATA was added to the sera to determine the concentration necessary to protect the RBCs from lysis by the zymosan-activated sera.

Results

We found that erythrocytes from PNH patients on long term treatment with eculizumab were twice as vulnerable as normal erythrocytes to lysis induced by complement activated serum. Western blot data showed the presence of both C3 and C5 convertases on the PNH patient erythrocyte membranes. These data indicate persistent vulnerability of PNH erythrocytes to complement attack due to deficiencies in CD55 and CD59. ATA, when added to serum in vitro, protected PNH erythrocytes from complement attack, restoring their resistance to that of normal erythrocytes.

Conclusions

We conclude that ATA, by protecting PNH erythrocytes from their decay accelerating factor (CD55) and protectin (CD59) deficiencies, may be an effective oral treatment in this disorder.     

Immunophenotyping and activation status of maternal peripheral blood leukocytes during pregnancy and labour,both term and preterm

The onset of labour in rodents and in humans is associated with physiological inflammation which is manifested by infiltration of activated maternal peripheral leukocytes (mPLs) into uterine tissues. Here, we used flow cytometry to immunophenotype mPLs throughout gestation and labour, both term and preterm. Peripheral blood was collected from non‐pregnant women and pregnant women in the 1st, 2nd and 3rd trimesters. Samples were also collected from women in active labour at term (TL) or preterm (PTL) and compared with women term not‐in‐labour (TNIL) and preterm not‐in‐labour (PTNIL). Different leukocyte populations were identified by surface markers such as CD45, CD14, CD15, CD3, CD4, CD8, CD19 and CD56. Their activation status was measured by the expression levels of CD11b, CD44, CD55, CD181 and CD192 proteins. Of all circulating CD45+ leukocytes, we detected significant increases in CD15+ granulocytes (i) in pregnant women versus non‐pregnant; (ii) in TL women versus TNIL and versus pregnant women in the 1st/2nd/3rd trimester; (iii) in PTL women versus PTNIL. TL was characterized by (iv) increased expressions of CD11b, CD55 and CD192 on granulocytes; (v) increased mean fluorescent intensity (MFI) of CD55 and CD192 on monocytes; (vi) increased CD44 MFI on CD3+ lymphocytes as compared to late gestation. In summary, we have identified sub‐populations of mPLs that are specifically activated in association with gestation (granulocytes) or with the onset of labour (granulocytes, monocytes and lymphocytes). Additionally, beta regression analysis created a set of reference values to rank this association between immune markers of pregnancy and to identify activation status with potential prognostic and diagnostic capability.     

Comparative analysis of different flow cytometry-based immunophenotypic methods for the analysis of CD59 and CD55 expression on major peripheral blood cell subsets

BACKGROUND: Flow cytometry-based immunophenotypic techniques for the analysis of CD55 and CD59 expression on the major cell populations present in blood are the preferred method for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH). METHODS: In the present study, we comparatively analyze the effects of stain-lyse-and-then-wash techniques and lyse-wash-and-then-stain procedures on the detection of both CD55 and CD59 expression on the major peripheral blood (PB) leucocyte subsets, as analyzed by flow cytometry. Our major goal was to establish the minimum amounts of anti-CD55 and anti-CD59 reagents required to be added to a minimum volume of blood, which would allow an optimal staining for both antigens on red cells, platelets, and leucocytes present in a single tube. RESULTS: Our results show that upon comparing stain-lyse-and-then-wash techniques with lyse-wash-and-then-stain protocols, the presence of important amounts of red cells at the time peripheral blood leucocytes are stained for CD55 and CD59 is associated with a significantly (P < 0.01) lower and more heterogeneous pattern of antigen expression on almost all major PB leucocyte subsets, supporting the need to use red cell lysing procedures prior to the staining of leucocytes. Identical, optimal patterns of antigen staining for CD55 and CD59 were obtained upon incubating 3 microL of blood with 10 microL of each of these monoclonal antibody (mAb) reagents (protein concentration of 0.05 microg/microL and 0.2 microg/microL respectively) for 30 min (room temperature [RT]) using a non-lyse-non-wash sample preparation procedure. This latter procedure allowed for the simultaneous analysis of CD55 and CD59 expression on red cells, platelets, neutrophils, monocytes, and lymphocytes present in the sample through the combined staining of CD55 and CD59 with CD64-fluorescein isothiocyante (FITC) plus CD61-peridinin chlorophyll protein (PerCP) and CD45-PerCP. CONCLUSIONS: In summary, our results show that the sample preparation protocol has a significant impact on the quality of the staining obtained for the CD55 and CD59 antigens on the major PB leucocyte subsets; additionally, we propose a simple and reliable stain-non-lyse-non-wash method for the simultaneous analysis of CD55 and CD59 expression on PB red cells, platelets, neutrophils, monocytes, and lymphocytes, which could be reached through the use of two triple stainings.     

H19, a surface membrane molecule involved in T-cell activation, inhibits channel formation by human complement.

Here we compare the properties of leukocyte antigens H19 and CD59 with those of the PI-linked 18,000-20,000 Mr molecules which inhibit lysis of human cells by the autologous terminal complement components C5b-9. H19, a 19,000 Mr protein found on human erythrocytes, monocytes, neutrophils, T-lymphocytes and other cells, is one of the ligands involved in the spontaneous rosette formation between human T-lymphocytes and erythrocytes. Recent evidence indicates that H19 also participates in T-cell activation. CD59 is a widely distributed 18,000-25,000 Mr protein anchored to the cell membrane by phosphatidylinositol (PI). The function of CD59 is unknown. Affinity-purified H19 incorporates into cell membranes and inhibits channel formation by human C5b-9 on guinea pig erythrocytes. Significant inhibition is achieved with picogram quantities of H19, corresponding to approximately 600 molecules per erythrocyte. H19 is most effective when C9 is limiting but quite active when C5b-7 or C8 are limiting, indicating that it may interact with several of the structurally related terminal complement components. The inhibitory activity is blocked by mAbs to either CD59 or to H19. H19 is PI-anchored: it is released from the cell membrane by treatment with PI-specific phospholipase C, and it is absent from cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). Analysis of PNH erythrocytes after treatment with terminal complement proteins shows that the H19-negative erythrocytes are more susceptible to C5b-9-mediated lysis. Treatment of normal human erythrocytes with either anti-H19 or anti-CD59 renders them more susceptible to lysis by human C5b-9. We conclude that H19 and CD59 are probably the same molecule and are identical or closely related to the recently described inhibitors of C5b-9 channel formation.     

L3T4(CD4)-, Lyt-2(CD8)- and Mac-1(CD11b)-phenotypic leukocytes in murine cryptococcal meningoencephalitis

An immunohistological study of L3T4(CD4)+ and LYT-2(CD8)+ lymphocytes, Mac-1(CD11b)+ monocytes and granulocytes in experimental murine cryptococcal meningoencephalitis was conducted. To assess the concomitant inflammatory reaction in an extracerebral site, livers were examined in parallel. Mice were infected i.v. withCryptococcus neoformans, group A/D, and organs were examined immunohistologically for CD4-, CD8- and monocyteand granulocyte-specific CD11b-phenotypic leukocytes over a period of 60 days. Intracerebrally, agglomerations of cryptococci formed pseudocysts that were surrounded by CD4+ and CD8+ lymphocytes at the end of the second week post-infection, followed by the invasion of monocytes and granulocytes into the lesions. After the fourth week post-infection, most of the invaded lesions were transformed into glious scars. Meningitis was usually marked and showed a homogenous distribution of CD4-, CD8- and CD11b-phenotypic cells, with a predominance of monocytes and CD4+ lymphocytes. Inflammatory infiltrates in the liver were found already 4 days post-infection. CD4+ lymphocytes and monocytes were distributed homogenously in the infiltrates, with a lower number of CD8+ lymphocytes being located rather in the periphery of the infiltrates. Comparing leukocyte kinetics in brain and liver, an important observation was the delayed immigration of immune cells at the intracerebral cryptococcal lesions as compared with the liver, and the different migration patterns of T-lymphocyte subgroups and macrophages. These results suggest that there are differential leukocyte migration patterns in the liver and brain following disseminated cryptococcosis. The immunological aspects of the observed leukocyte kinetics are discussed.     

Species differences in the expression of carbohydrate differentiation antigens on mammalian blood cells revealed by immunofluorescence with monoclonal antibodies

Following recent observations using monoclonal antibodies that carbohydrate structures behave as differentiation antigens of man and mouse, we have made a preliminary survey of the expression of 8 monoclonal antibody-defined carbohydrate antigens on blood cell smears of man, baboon, mouse, rat, rabbit, pig, and dog. There are considerable species differ-ences in the patterns of antigen expression. However, certain generalizations can be made as follows: the i and I antigens, associated with linear and branched carbohydrate chains consisting of repeating N-acetyl-lactosamine sequences (GalB1-4GIcNAc, termed Type-2 backbone sequences) are widely distributed among granulocytes and lymphocytes of all the species studied, and on erythrocytes, monocytes, and piateiets of some of them. Substantial amounts of Type-1 backbone sequences (GalB1-3GlcNAc) may occur on rabbit lymphocytes. The N-acetylneuraminic acid-containing antigens, Pr 2 and Gd, are also expressed to varying degrees on blood cells. On the other hand, antigens based on mono- and diiucosylated N-acetyllactosamine, termed SSEA-1 (or X-hapten) and C14 (or Y-hapten) are predominantly granulocyte/monocyte-associated antigens. The former antigen is expressed in overt form only on untreated human granulocytes but occurs in cryptic state , masked by sialic acid, on human monocytes, and on the granulocytes and monocytes of baboon, rabbit, and dog but not on those of mouse, rat, and pig. The latter antigen is expressed on human granulocytes and on neuraminidase-treated monocytes and granulocytes oi dog. Lymphocytes of dog are unusual in their expression of C14 antigen, in cryptic state, masked by sialic acid residues. Although the physiological roles of these various carbohydrate structures , in vivo, are not yet known, they seem excellent candidates as determinants of species and cell-type differences in susceptibilities to infective agents.     

Analysis of heparin binding to human leukocytes using a fluorescein-5-isothiocyanate labeled heparin fragment

The binding of LMWH-tyr-FITC to granulocytes, monocytes, and lymphocytes was analyzed by flow cytometry using a low-molecular-weight heparin (LMWH) labeled with fluorescein-5-isothiocyanate (FITC). FITC was covalently bound to tyramine, which was synthesized to LMWH by endpoint-attachment (Malsch et al.: Anal Biochem 217:255-264, 1994). The binding was rapid, specific, dose-dependent, saturable, and reversible. To investigate the molecular weight dependence of heparins, heparin-derived di- to dodecasaccharides were used. With decreasing molecular weight, the amount of oligosaccharides increased; these were bound to granulocytes, monocytes, and lymphocytes (r = -0.77). The degree of sulfation of non-heparin glycosaminoglycans influenced the binding to leukocytes. Decreasing the degree of sulfation decreased the binding. The pentasaccharide did not bind as strongly as the other heparin-derived oligosaccharides, indicating an AT III-independent mechanism. Two classes of heparin binding sites were identified on granulocytes and one class of binding sites on monocytes and lymphocytes. The lowest amount of LMWH-tyr-FITC detected was 1 ng on granulocytes, 0.18 ng on monocytes and 0.01 ng on lymphocytes. The data suggest that heparin and other sulfated polysaccharides may play a role in the physiology of thrombosis, arteriosclerosis, and inflammation by binding to granulocytes, monocytes, and lymphocytes.     

The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection.

The bovine leukemia virus (BLV) is an oncogenic retrovirus that is associated with the development of persistent lymphocytosis (PL) and lymphoma in cattle. While B lymphocytes have been shown to be the primary cellular target of BLV, recent studies suggest that some T lymphocytes and monocytes may be infected by the virus. Because virally altered functions of monocytes and/or T cells could contribute to the development of lymphoproliferative disease, we sought to clarify the distribution of the BLV provirus in subpopulations of peripheral blood mononuclear cells in seropositive cows with and without PL. CD2+ T cells, monocytes, and CD5+ and CD5- B cells were sorted by flow cytometry and tested for the presence of BLV by single-cell PCR. We did not obtain convincing evidence that peripheral blood monocytes or T lymphocytes contain the BLV provirus in seropositive cows with or without PL. In seropositive cows without PL (n=14), BLV-infected CD5+ and CD5- B cells accounted for 9.2% +/- 19% and 0.1% +/- 1.8% of circulating B lymphocytes, respectively. In cows with PL (n=5), BLV-infected CD5+ and CD5- B cells accounted for 66% +/- 4.8% and 13.9% +/- 6.6% of circulating B lymphocytes, respectively. The increase in lymphocyte numbers in cows with PL was entirely attributable to the 45-fold and 99-fold expansions of infected CD5+ and CD5- B-cell populations, respectively. Our results demonstrate that B cells are the only mononuclear cells in peripheral blood that are significantly infected with BLV. On the basis of the absolute numbers of infected cells in seropositive, hematologically normal animals, there appear to be differences in susceptibility to viral spread in vivo that may be under the genetic control of the host.     

Human blood dendritic cell-like B cells isolated by the 5G9 monoclonal antibody reactive with a novel 220-kDa antigen.

We developed a murine IgG1 mAb, 5G9, following immunization of a BALB/c mouse with Daudi cells. By immunoprecipitation, 5G9 reacted with a 220-kDa Ag on Daudi cells, which reduced to four subunits (55, 65, 80, and 85 kDa). mAb 5G9 bound to 40-60% of peripheral blood B cells, weakly reacted with monocytes and granulocytes, and did not bind to erythrocytes, platelets, T cells, or NK cells. mAb 5G9 brightly stained scattered cells in human tonsil sections, which appeared to be dendritic cells (DC) by morphology. mAb 5G9 also stained scattered cells in cytospin slides of monocyte-derived DC with long, thin, beaded membrane processes, morphologically distinct from other monocyte-derived DC. Positive selection of blood mononuclear cells with mAb 5G9 and sheep anti-mouse IgG Dynabeads demonstrated an enriched population of DC. By flow cytometry analysis, these cells were CD19, CD20, CD22, CD40, CD44, CD83, CD86, IgD, and HLA-Dr positive and either kappa- or lambda-L chain positive. They did not express CD3, CD4, CD5, CD10, CD11b, CD13, CD25, CD56, CD14, CD33, or CD64. Isolated 5G9+ cells were potent APCs in allogeneic MLR, compared with 5G9- PBMC, 5G9- B cells, monocytes, and monocytes cultured in IL-4 and GM-CSF for 24 h. mAb 5G9 defines a novel peripheral blood cell with B cell phenotype and DC morphology and function: DC-like B cells. The significance of this cell in immune responses requires further study.     

Natural killer cells are deficient in the surface expression of the complement regulatory protein, decay accelerating factor (DAF)

Decay-accelerating factor (DAF) is a 75,000 m.w. membrane protein that inhibits autologous complement C3 activation at the cell surface. One-color direct immunofluorescence with anti-DAF antibody and cytofluorographic analysis indicates that normal human monocytes and granulocytes are uniform in expression of DAF, whereas 23% of peripheral blood lymphocytes are DAF deficient. A two-color indirect immunofluorescence method, used to define the phenotype(s) of the DAF-deficient lymphocytes, was less efficient in DAF detection and led to overestimation of the fraction of deficient cells. Nonetheless, the difference between DAF expression by natural killer cells, identified by the CD16 and HNK-1 antigens, was marked. DAF deficiency was intermediate in cells expressing the CD2, CD3, CD4, or CD8 markers. On the basis of the phenotypic definition of natural killer cells and their contribution to the lymphocyte population, it is concluded that a uniform deficiency of DAF on natural killer cells accounts for about one-half of the DAF-deficient lymphocytes in peripheral blood of normal donors. The finding of a complete DAF deficiency in the lymphocytes from a patient with a lymphoproliferative disorder with the predominant proliferation of CD2+, CD3+, CD8+, HNK-1+ large granular lymphocytes gives additional support for the association of DAF-deficiency with natural killer cells.     

Expression of endothelial selectin ligands on human leukocytes following dive

The fact that impaired endothelial-dependent vasodilatation after scuba diving often occurs without visible changes in the endothelial layer implies its biochemical origin. Since Lewis x (CD15) and sialyl-Lewis x (CD15s) are granulocyte and monocyte carbohydrate antigens recognized as ligands by endothelial selectins, we assumed that they could be sensitive markers for impaired vasodilatation following diving. Using flow cytometry, we determined the CD15 and CD15s peripheral blood mononuclear cells of eight divers, 30 mins before and 50 mins after a single dive to 54 m for 20 mins bottom time. The number of gas bubbles in the right heart was monitored by ultrasound. Gas bubbles were seen in all eight divers, with the average number of bubbles/cm(2) 1.9 +/- 1.9. The proportion of CD15 + monocytes increased 2-fold after the dive as well as the subpopulation of monocytes highly expressing CD15s. The absolute number of monocytes was slightly, but not significantly, increased after the dive, whereas the absolute number of granulocytes was markedly elevated (up to 61%). There were no significant correlations between bubble formation and CD15 + monocyte expression (r = - 0.56; P = 0.17), as well as with monocytes highly expressing CD15s (r = 0.43; P = 0.29). This study suggests that biochemical changes induced by scuba diving primarily activate existing monocytes rather than increase the number of monocytes at a time of acute arterial endothelial dysfunction.     

CD14 is a component of multiple recognition systems used by macrophages to phagocytose apoptotic lymphocytes.

Expression of the aminophospholipid phosphatidylserine (PS) on the surface of apoptotic lymphocytes and lipid-symmetric erythrocytes triggers their phagocytosis by macrophages. Phagocytosis by both activated and unactivated macrophages, which utilize different recognition systems, can be blocked by certain monoclonal antibodies directed against the LPS receptor, CD14. Here we investigate the requirement for CD14 in the phagocytosis of both apoptotic thymocytes and lipid-symmetric erythrocytes by both activated and unactivated macrophages. We show that phagocytosis of lipid-symmetric erythrocytes by both activated and unactivated macrophages is completely abolished when CD14 is removed from macrophages by cleaving its glycosylphosphatidylinositol tether with phospholipase C. This treatment also substantially reduces phagocytosis of apoptotic lymphocytes by both types of macrophages. Unactivated LR-9 mouse macrophages which are deficient in CD14 expression are completely unable to phagocytose either apoptotic thymocytes or lipid-symmetric erythrocytes. These results argue that CD14 is an absolute requirement for the phagocytosis of lipid-symmetric erythrocytes by both activated and unactivated macrophages, despite their different recognition systems, that CD14 contributes at least substantially to the phagocytosis of apoptotic lymphocytes by both activated and unactivated macrophages, and that activated macrophages may also possess an alternate, CD14-independent mechanism for phagocytosis of apoptotic lymphocytes.     

Increased lymphocytic aminopeptidase N/CD13 promoter activity after cell-cell contact

Aminopeptidase N (APN)/CD13 is a transmembrane ectoenzyme expressed on a wide variety of cells. With respect to haematopoietic cells, APN/CD13 has been considered specific for the myeloid lineage, because granulocytes and monocytes/macrophages, but not lymphocytes of peripheral blood, show a surface expression of CD13 antigen. However, we could recently show that cell-cell contact of lymphocytes with endothelial cells, monocytes, and fibroblast-like synoviocytes (SFCs) results in an increase of steady-state APN/CD13 mRNA and a rapid expression of cell-surface protein on the lymphocytes. In this study using the Dual-Luciferase reporter assay, we demonstrate that interaction of the T-cell line Jurkat with SFCs results in a higher activity of the APN/CD13 myeloid promoter in T cells. An enhancer located between the myeloid and epithelial APN/CD13 promoter increases the response of the promoter to the cell-cell contact-induced expression of APN/CD13 in lymphocytes. Adhesion of lymphocytes to extracellular matrix did not result in increased promoter activity. The lymphocytic promoter response induced by direct cell-cell contact with SFCs is not affected by mutations of a proximal promoter element (nucleotides -48 to -35), which has a possible functional role in the basal APN/CD13 gene expression in lymphocytes. Upregulated peptidase-promoter activity via cell-cell contact shown in this study for the first time is discussed as a general mechanism in peptidase induction.     

Ly-6G+CCR2- myeloid cells rather than Ly-6ChighCCR2+ monocytes are required for the control of bacterial infection in the central nervous system

Myeloid cell recruitment is a characteristic feature of bacterial meningitis. However, the cellular mechanisms important for the control of Streptococcus pneumoniae infection remain largely undefined. Previous pharmacological or genetic studies broadly depleted many myeloid cell types within the meninges, which did not allow defining the function of specific myeloid subsets. Herein we show that besides CD11b(+)Ly-6G(+)CCR2(-) granulocytes, also CD11b(+)Ly-6C(high)CCR2(+) but not Ly-6C(low)CCR2(-) monocytes were recruited in high numbers to the brain as early as 12 h after bacterial challenge. Surprisingly, CD11b(+)Ly-6C(high)CCR2(+) inflammatory monocytes modulated local CXCL2 and IL-1beta production within the meninges but did not provide protection against bacterial infection. Consistent with these results, CCR2 deficiency strongly impaired monocyte recruitment to the infected brains but was redundant for disease pathogenesis. In contrast, specific depletion of polymorphonuclear granulocytes caused elevated local bacterial titer within the brains, led to an aggravated clinical course, and enhanced mortality. These findings demonstrate that Ly-6C(high)CCR2(+) inflammatory monocytes play a redundant role for the host defense during bacterial meningitis and that predominantly CD11b(+)Ly-6G(+)CCR2(-) myeloid cells are involved in the restriction of the extracellular bacteria.     

Electron tomography of negatively stained complex viruses: application in their diagnosis

Pattern recognition receptors are a key component of the first line host defense against infection, recognizing specific microbial products. We hypothesize that monocyte hyporesponsiveness in human sepsis is associated with a downregulation of the pattern recognition receptors Toll-like receptor (TLR)-2 and TLR4. Protein expression of CD14, TLR2 and TLR4 on blood monocytes was examined using flow cytometry from 29 patients with sepsis and 14 healthy controls. In addition LPS stimulated TNF-α and IL-10 production was studied in a 24 hour whole blood assay. We found an increased expression of CD14, TLR2 and TLR4 in patients with sepsis compared to controls (p < 0.01). In patients with sepsis, death was associated with significant lower CD14 and TLR2 expression at admission (CD14: 25.7 +- 19.1 vs 39.1 +- 17.3 mean fluorescence intensity [MFI], p = 0.02; TLR2: 21.8 +- 9.4 vs. 30.9 +- 9.6, p = 0.01). At 72 hours the TLR2 expression on monocytes was associated with the IL-10 inducibility after LPS stimulation (r = 0.52, p = 0.02) and the CD14 expression with the IL-6, IL-10 and TNF inducibility. We conclude that septic patients are characterized by an increased expression of CD14, TLR2 and TLR4 on monocytes compared to controls. Death is associated with downregulation of TLR2 and CD14 expression on monocytes correlating with reduced cytokine inducibility. We suggest that CD14 and TLR2 are a key factor in monocyte hyporesponsibility during severe sepsis.     

Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells

Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood cells isolated from healthy volunteers by density centrifugation and elutriation centrifugation. We also investigated the mRNA expression of CTP synthase in lymphocytes and monocytes. The highest activity of CTP synthase was found in thrombocytes (6.48 nmol CTP x mg(-1) x h(-1)), followed by that of monocytes (2.23), lymphocytes (1.69), granulocytes (0.52) and erythrocytes (0.42). The activity of CTP synthase in whole blood samples was at an intermediate level (1.27). The mRNA expression of CTP synthase in monocytes was comparable to that observed in lymphocytes.