Prostaglandin E2, monocyte adherence and interleukin-1 in the regulation of human natural killer cell activity by monocytes

Monocytes have previously been shown both to augment and suppress human natural killer (NK) cell activity depending upon the conditions. An interleukin-1/interleukin-2 (IL-1/IL-2)-dependent mechanism has been shown to be involved in the augmentative effect. In the current study, the role of the method of monocyte isolation was evaluated. Monocytes isolated by Percoll gradient centrifugation were ineffective for modulating NK activity, but monocytes isolated by adherence from most donors exhibited increased augmentation with increased interval of adherence (up to 1 h). However, monocytes isolated by adherence from certain donors reproducibly exhibited increased suppression with increased interval of adherence. The observation of augmentation was correlated with an increase in the balance between IL-1 production and prostaglandin E (PGE) production by the monocytes. The roles of PGE2 and IL-1 were therefore examined by mixing these cytokines with enriched null lymphocyte preparations in the absence or presence of monocytes in the NK assay system. The participation of PGE2 was further examined using monocytes treated with indomethacin (10(-6) M), and the participation of monocyte-membrane-bound IL-1 was evaluated using monocytes fixed with 1% paraformaldehyde. The results revealed that PGE2 production is involved in the suppression of human NK activity by human monocytes, and the functional balance between IL-1 and PGE2 determines whether suppression or augmentation is observed. The data of this and previous studies are consistent with the suggestion that membrane-associated IL-1 is the important IL-1 moiety for the augmentation of human NK activity by monocytes.     

Accessory cell requirements for the mixed-leukocyte reaction and polyclonal mitogens, as studied with a new technique for enriching blood dendritic cells

Human blood dendritic cells can be enriched to 40-80% purity by a new technique that is simpler, provides greater yields than prior methods, and resolves other populations that are enriched in monocytes and B and T lymphocytes. The procedure involves separation over two Percoll gradients after 0 and 2 days of culture, followed by removal of contaminating monocytes by panning on plates coated with human Ig. The resultant dendritic cell-enriched fraction is 10 times or more potent than the monocyte-enriched populations in stimulating T-cell proliferative responses to alloantigens and to Con A. Small B lymphocytes are inactive in both systems. Dendritic cells do not initiate mitogenesis to anti-CD3 monoclonal antibodies, a response for which the monocyte appears to be the critical accessory cell.     

Separation of lymphokine-activated killer cell precursors and T-cells: differential growth characteristics and apparent lack of a T-cell suppressor of LAK-cell induction

Lymphokine activated killer (LAK) cell clinical effectiveness may be limited by the total cell dose and cytotoxic activity. We have, therefore, examined methods to expand the number of LAK-cells by serial passage of unfractionated and fractionated peripheral blood lymphocytes. Human purified lymphocytes were obtained by Ficoll Hypaque gradients followed by exposure of resultant mononuclear cells to phenylalanine methyl ester to remove monocytes. Lymphocytes were then fractionated on a six-step Percoll gradient (50%, 47.5%, 45%, 42.5%, 40%, and 37.5% Percoll). Unfractionated cells and fractions were cultured in standard media (RPMI-1640, 10% human sera, antibiotics and 10 mM HEPES) containing 10 nM of recombinant Interleukin-2 (rIL-2). Lymphocytes were cultured at 1 X 10(6)/ml and recultured every 3 to 4 days in fresh standard media and rIL-2. Utilizing unfractionated and fractionated lymphocytes from seven donors we made the following observations: (1) Continued passage of unfractionated lymphocytes resulted in a loss of LAK-cell activity by greater than or equal to 14 days (e.g., percent lysis of Raji at 10:1 effector:target ratio on days 0, 4, 7, and 21 was 0.38 +/- 11, 41 +/- 17, and 8 +/- 1, n = 4, respectively). (2) LAK-cell functional precursors were predominantly confined to the lymphocytes in the upper (0-4) Percoll fractions (e.g., on day 4, the percent lysis by pooled fractions 1-4 was 63 +/- 5 vs. pooled fraction 5 plus pellet, 18 +/- 7%). (3) As expected, the upper fractions (0-4) were enriched for Leu 19 positive cells (approximately 40%) and large granular lymphocytes (LGL) by morphology (approximately 30%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Phagocytosis by catfish neutrophils

Channel catfish peripheral blood leucocytes were separated on a Percoll gradient to establish the phagocytic function of the neutrophils. Four fractions of leucocytes were formed on the Percoll gradient, including a fraction that contained 50–80% neutrophils at a density of 1.08–1.09 g ml⁻¹ and a fraction that contained 10% monocytes at a density of 1.071–1.074 g ml⁻¹. Phagocytic assays, using ³H-uridine, showed that the two fractions had similar phagocytic indices, although neutrophils were less phagocytic than monocytes. Neutrophils were confirmed to be phagocytic when examined with transmission electron microscopy. Staining with 3,3-diaminobenzidine-tetrahydrochloride demonstrated peroxidase-positive granules in the cytoplasm of actively phagocytic cells as well as peroxidase reaction products in a number of phagosomes containing bacteria. Phagocytosis of bacteria by channel catfish neutrophils was further confirmed by differential staining of external bacteria and cell surfaces with ruthenium red during the fixation process.     

Augmentation of human monocyte-mediated cytolysis by interferon

Human monocytes, separated by either plastic adherence or adherence to microexudatecoated surfaces, from the peripheral blood of most normal donors were shown to have significant cytolytic activity against TU5, a mouse SV40-transformed target cell. Spontaneous cytolysis ranged from 0 to 32% at a 40:1 effector:target (E:T) ratio. Augmentation of cytolysis was usually seen when human fibroblast interferon (IF) (10³–10⁴ units/ml) was cultured with the effector and target cells for the duration of the assay. The mean increase in percentage cytolysis at 40:1 and 20:1 E:T ratios was greater with monocytes obtained by a microexudate method (24.1 and 22.4%) than with monocytes obtained by a plastic adherence method (16.0 and 8.1%). Only a slight augmentation of cytotoxicity was observed when the effector cells were pretreated with IF for 1-hr. The increased levels of cytotoxicity observed when IF was present during the assay did not appear to be due to the toxic effects of IF on the target cells or to a stable increase in the susceptibility of the target cells to lysis.     

Integrated morphophysiological assessment of two methods for sperm selection in bovine embryo production in vitro

Extensive work was done regarding the ability of Swim up and Percoll gradient to select functional sperm for in vitro embryo production (IVP) systems. The aim of this work was to compare Swim up and Percoll as methods of sperm selection by ultrastructural, biochemical and functional studies. Frozen-thawed semen from two bulls (Experiments 1 and 2, respectively) were treated using Swim up or Percoll discontinuous gradients. Motility, sperm membrane ultrastructure, sperm proteins, in vitro embryo production (insemination doses, cleavage, embryo yield and quality) and embryo sex ratio were scored and compared. Electron transmission microscopy of outer sperm membranes showed higher (P<0.05) percentage of sperm with lost acrosomes in Percoll treated samples compared to Swim up. A differential protein pattern was also detected. When in vitro embryo production was performed, Percoll gradient produced higher (P<0.05) number of fertilizing doses (7.6 versus 5.9, Bull 1; 13.5 versus 7.8, Bull 2) and higher sperm motility (90% versus 76.6%, Bull 1; 81.7% versus 68.3%, Bull 2) than Swim up. The percentage of cleavage (Day 3) was similar in both treatment groups, whereas embryo production rate (Day 7) was higher (39.4% versus 30.2%, Bull 1; 38% versus 32.4%, Bull 2; P<0.05) when Percoll gradient was used. The percentage of hatched embryos (Day 11) and sex ratio did not differ. Total cell counting and embryo differential staining (inner cell mass and trophoblast cells) of Day 7 embryos showed that Percoll treated sperm produced better quality embryos compared to Swim up. We concluded that Percoll had a better performance selecting sperm and an enhanced capacity for embryo production when compared with the Swim up procedure; this could be attributed to a better acrosome exocytosis, associated to the absence of certain membrane proteins.     

Characterization of monocyte adherence to human macrovascular and microvascular endothelial cells

The interaction of monocytes with cultured large vessel venous and arterial endothelial cells (EC) and with cultured microvascular EC was studied. Analysis of time-lapse microcinematographic video recordings showed that monocytes adhere rapidly to the surface of EC and subsequently remain spherical and fixed to the initial site of adherence. Some monocytes adherent to EC stretch out within 30 to 90 min and migrate over the EC surface or become stretched for about 10 to 30 min and then detach from the EC surface and move rapidly over the EC monolayer. It was shown that the interaction of monocytes with EC is dynamic, that the morphology of monocytes adherent to EC changes constantly, and that stretching of the monocytes over the surface of the EC is not an inevitable and irreversible consequence of binding. A quantitative adherence assay was developed in which both the morphology and the number of monocytes bound to EC were determined. For each type of EC the number of monocytes bound to a single EC was found to be linearly related to the number of monocytes added and was lower for smaller EC. The adherence of monocytes to venous and arterial EC followed a different time course than the adherence to capillary EC and adherence to both types of macrovascular EC was higher than adherence to microvascular EC was higher than adherence to microvascular EC. The percentage of adherent monocytes with a stretched morphology was lower when these cells were adherent to capillary EC than to both types of macrovascular EC and increased upon addition of serum. Adherence of monocytes to venous, arterial, and capillary EC was partially inhibited by mAb directed against the alpha-chain of lymphocyte function-associated Ag-1 or C3bi receptor (with mAb LM2/1, but not with mAb OKM1) and by mAb against the common beta-chain of the three leukocyte adhesion molecules. The degree of inhibition of monocyte adherence to EC by mAb against lymphocyte function-associated Ag-1 alpha and the common beta-chain was dependent on the type of EC and was higher for venous EC (57 to 70% inhibition) than for arterial (40 to 44% inhibition) and capillary (44 to 49% inhibition) EC. Inhibition of monocyte adherence obtained with anti-C3bi receptor-alpha mAb was similar for each EC type. mAb against p150, 95 did not affect adherence. None of the mAb could block binding completely; combinations of the mAb also did not result in increased inhibition of monocyte adherence to EC.     

Antioxidants improve antibacterial function in hyperoxia-exposed macrophages

Hyperoxia and pulmonary infections are well known to increase the risk of acute and chronic lung injury in newborn infants, but it is not clear whether hyperoxia directly increases the risk of pneumonia. The purpose of this study was to examine: (1) the effects of hyperoxia and antioxidant enzymes on inflammation and bacterial clearance in mononuclear cells and (2) developmental differences between adult and neonatal mononuclear cells in response to hyperoxia. Mouse macrophages were exposed to either room air or 95% O2 for 24 h and then incubated with Pseudomonas aeruginosa. After 1 h, bacterial adherence, phagocytosis, and macrophage inflammatory protein (MIP)-1alpha production were analyzed. Bacterial adherence increased 5.8-fold (p < 0.0001), phagocytosis decreased 60% (p < 0.05), and MIP-1alpha production increased 49% (p < 0.05) in response to hyperoxia. Overexpression of MnSOD or catalase significantly decreased bacterial adherence by 30.5%, but only MnSOD significantly improved bacterial phagocytosis and attenuated MIP-1alpha production. When monocytes from newborns and adults were exposed to hyperoxia, phagocytosis was impaired in both groups. However, adult monocytes were significantly more impaired than neonatal monocytes. Data indicate that hyperoxia significantly increases bacterial adherence while impairing function of mononuclear cells, with adult cells being more impaired than neonatal cells. MnSOD reduces bacterial adherence and inflammation and improves bacterial phagocytosis in mononuclear cells in response to hyperoxia, which should minimize the development of oxidant-induced lung injury as well as reducing nosocomial infections.     

Human monocyte adherence: a primary effect of chemotactic factors on the monocyte to stimulate adherence to human endothelium

Monocyte emigration into areas of inflammation is initiated by monocyte adherence to the microvascular endothelium which may be induced by the local production of chemotactic factors at the inflammatory site. However, it is not clear whether such stimuli act on the monocyte and/or the endothelial cell to promote this effect. Accordingly, the effect of the chemotactic peptides C5a des arg and formyl-methionyl-leucyl-phenylalanine (FMLP) on human monocyte adherence to human microvascular endothelial cell monolayers was investigated in vitro. Monocytes (92 to 98% pure) were isolated by discontinuous plasma-Percoll density gradients and cell elutriation, methods designed to minimize monocyte exposure to endotoxin. Mean spontaneous (unstimulated) adherence of 111Indium-tropolonate-radiolabeled monocytes to microvascular endothelial cell monolayers was 19.7% +/- 1.3. Monocyte adherence to microvascular endothelial cell monolayers was stimulated in a dose-response fashion in the presence of C5a des arg or FMLP to a maximum mean adherence of 47.2% +/- 2.9 or 43.8% +/- 2.2, respectively. C5a des arg or FMLP stimulated monocytes to adhere to monolayers of human vascular smooth muscle cells, human dermal fibroblasts, or serum-coated plastic wells in a comparable fashion as to endothelial cells. The simultaneous presence of both chemotactic peptides C5a des arg and FMLP in the assay system stimulated monocyte adherence to the same degree as either stimulus alone. This finding suggested that those monocytes stimulated to adhere by C5a des arg were the same subpopulation responding to FMLP. Spontaneous monocyte adherence (in the absence of chemotactic peptides) to both endothelial cell monolayers and serum-coated plastic wells was reduced in the presence of plasma, but chemotactic peptides induced a significant, albeit reduced, adhesion of monocytes in this circumstance. The pretreatment of monocytes with either C5a des arg or FMLP prior to the adherence assay induced stimulus-specific desensitization of monocyte adherence. Neither a desensitization nor stimulated monocyte adherence occurred when endothelial cell monolayers or serum-coated plastic wells were pretreated with either of the chemotactic peptides. The fixation of endothelial cell monolayers prior to the adherence assay did not alter the degree of spontaneous, C5a des arg-stimulated, or FMLP-stimulated monocyte adherence. These data suggest that the stimulated adhesion of monocytes to endothelial cells by C5a des arg or FMLP represents primarily an effect of these chemotactic peptides on the monocyte.     

Mechanism of cigarette smoke condensate induced adhesion of human monocytes to cultured endothelial cells

Cigarette smoking is ranked among the leading risk factors in the etiology of atherosclerotic vascular disease. The mechanisms, however, that link cigarette smoking to increased incidence of atherosclerosis are not understood. The adherence of circulating monocytes to the endothelium, migration into the subendothelium, and subsequent formation of foam cells are principal initial events in the development of atherosclerosis. We therefore determined whether cigarette smoke caused increased adherence of monocytes to endothelial cells and the cellular mechanism of this increased adherence. Cigrette smoke condensate (CSC), the particulate fraction of cigarette smoke derived from 2R1 standard research cigarettes, at a concentration of 25–30 μg/ml (average yield of CSC is 26.1 mg/cigarette), augmented (70–90%) basal adherence of human peripheral blood monocytes to a cultured monolayer of endothelial cells derived from bovine aorta (BAEC) and human umbilical vein (HUVEC). There was a concomitant increase in the expression of CD11b ligand on the surface of monocytes as determined by flow cytometry, utilizing FITC conjugated Mab MO-1 (CD11b). However, nicotine (1–15 μg/ml) and cadmium sulfate (10 μg/ml), constituents of CSC, individually or in combination had no effect either on CD11b expression or adherence of monocytes to endothelial cells. Treatment of HUVEC with CSC for 60 min also resulted in an increased expression of ICAM-1 and ELAM-1 as determined by mean fluorescence intensity of ICAM-1 and ELAM-1 labeled cells in flow cytometric analysis. The CSC induced expression of CD11b in monocytes was optimal at 25–30 min and was inhibited by protein kinase C inhibitors, staurosporine and H-7, and also by baicalein, a lipoxygenase inhibitor. Similarly, CSC induced ICAM-1 and ELAM-1 expression in HUVEC was inhibited by protein kinase C inhibitors. CSC stimulated the adherence of human monocytes but not the monocytic cell lines HL-60, U937, and THP-1 to endothelial cells. The CSC stimulated adherence of human monocytes was inhibited (80%) by MAb to CD11b and 50% by Mab to ICAM-1 and ELAM-1. These results suggest that cigarettee smoke particulate constituents activate protein kinase C, leading to increased surface expression of adhesive ligand CD11b on peripheral blood monocytes and counter receptor(s) ICAM-1 and ELAM-1 in endothelial cells. The expression of ligand and counter receptor leads to potentiated adherence of monocytes to endothelial cells, an initial event in the pathogenesis of cigarette smoke induced inflammatory response in the vessel wall. © 1994 Wiley-Liss, Inc.     

人早孕胎盘绒毛膜滋养层细胞体外培养模型的建立

目的通过对早孕胎盘绒毛膜滋养层细胞的分离,纯化和培养,寻找一种稳定、简便可获得较高纯度滋养层细胞的培养方法。方法通过胰酶/DNA酶联合消化法对妊娠6-10周绒毛组织进行消化,获得单细胞悬液,比较Per-coll密度梯度离心和淋巴细胞分离液对滋养层细胞的分离纯化效果。含10?S的DMEM/F12培养基培养,并比较是否应用鼠尾胶原对细胞贴壁和生长的影响。通过免疫荧光方法对滋养层细胞进行鉴定。结果经简化Percoll密度梯度离心分离纯化的滋养层细胞纯度高,明显优于淋巴细胞分离液的分离效果(P<0.001);细胞生长表面预先经鼠尾胶原处理后,细胞贴壁良好,分裂生长旺盛。结论利用简化Percoll密度梯度离心法分离细胞,并在应用鼠尾胶原的条件下进行培养,可以获得满意的人绒毛膜滋养层细胞的体外培养模型。     

Lipopolysaccharide exposure during purification of human monocytes by adherence increases their recovery and cytolytic activity

Exposure of monocytes to lipopolysaccharide (LPS) during, but not after, adherence purification increased their cytolytic activity in short-term 51Cr-release assays against K562 target cells. In the absence of LPS only a minority of monocytes could be recovered by adherence. With 1 ng/ml to 10 micrograms/ml LPS present during the 1-hr adherence procedure, however, monocytes spread more extensively on serum-coated plastic and glass surfaces and virtually all of the monocytes in a mononuclear leukocyte preparation were recovered in the adherent fraction. While increasing the recovery of monocytes threefold, LPS exposure during adherence also increased monocyte purity as assessed by peroxidase staining, morphology, and indirect immunofluorescence with monoclonal Mo2. The proportion of Leu-11-positive NK cells in the adherent fraction did not change. Depletion of NK cells by treatment with anti-Leu-11b and complement eliminated cytolytic activity from the nonadherent, but not from the adherent, fraction isolated with LPS. Thus, addition of LPS during adherence produced a monocyte preparation with enhanced cytolytic activity not attributable to NK contaminants. To test whether LPS caused production of lymphokines that activate monocytes, we tested supernatants of unseparated mononuclear leukocytes for the capacity to stimulate purified monocytes for cytolysis. Such supernatants stimulated monocytes more effectively than LPS alone. We conclude that LPS stimulates monocytes for cytolysis most effectively during adherence purification because LPS allows the recovery of weakly adherent monocytes with high cytolytic capacity; also, LPS may stimulate production of lymphokines that further augment monocyte cytolytic activity.     

Synthesis of tumor necrosis factor-alpha (TNF-alpha) by human monocytes in vitro]

The presence of TNF-alpha mRNA and protein in circulating human blood monocytes isolated by continuous Percoll gradient fractionation was studied. The technique of RNA isolation from the blood samples was used to study TNF-alpha mRNA expression. It was shown that human blood monocytes of healthy donors contained no presynthesized pool of TNF-alpha mRNA as well as no TNF-alpha protein.     

Chemotaxis of human blood monocytes toward endothelin-1 and the influence of calcium channel blockers.

The adherence of monocytes to the arterial endothelium followed by its migration into the arterial intima is the earliest event in atherogenesis. The vasoconstrictive peptide, Endothelin-1 (ET-1), is elevated in patients with atherosclerosis. We were interested to know whether ET-1 was a chemoattractant for blood monocytes. Using the modified membrane filter technique for chemotaxsis assessment, ET-1 increased monocyte chemotaxis in a dose-dependent manner. Ca2+ channel blockers, Nifedipine, Diltiazem and Verapamil (5 microM), reduced ET-1 chemotaxsis more than 60% (P < 0.001). Aspirin and Indomethacin (1 mM and 100 microM, respectively) reduced migration by 23% (P < 0.05). Alpha-Lipoic acid, Probucol and Neomycin (100 microM) were also migration inhibitory (37%, P < 0.01). These results suggest that ET-1 is a strong chemoattractant for blood monocytes; Ca2+ influx is probably the major stimulus for the accelerated migration induced by ET-1.     

Contrasting effects of inflammatory stimuli on neutrophil and monocyte adherence to endothelial cells

Leukocyte adherence to endothelial cells (EC) is an important early event in inflammatory responses, which are often characterized by a predominance of either neutrophils (PMN) or monocytes. However, there is little information concerning the molecular events important in leukocyte adherence to EC. Intracellular activation of protein kinase C and the calcium-second messenger system leads to the stimulation of a number of important functions in PMN and monocytes. We compared the effects of members of these pathways on human PMN and monocyte adherence to cultured bovine aortic EC. We observed that phorbol myristate acetate, phorbol, 12,13-dibutyrate, L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol, and ionomycin each induced significant dose-dependent increases in PMN adherence to EC monolayers. In contrast, similar concentrations of each of these agents induced significant decreases in EC adherence of monocytes enriched by countercurrent centrifugal elutriation. Separate experiments determined that the differences in PMN and monocyte adherence to EC were not related to differences in oxidant production because 1) phorbol myristate acetate and L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol caused similar marked increases in both PMN and monocyte superoxide anion and hydrogen peroxide production and 2) ionomycin, which had opposing effects on PMN and monocyte adherence, had no effect on PMN and monocyte superoxide anion or hydrogen peroxide release. We conclude that activators of protein kinase C and the Ca-second messenger pathway have opposite effects on PMN and monocyte adherence to EC and that these effects are mediated by O2 radical-independent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of HLA-DR antigen expression in phytohemagglutinin-activated T cells in man. Requirement of T cell recognition of self HLA-DR antigen expressed on the surface of monocytes

Signals required for expression of HLA-DR (DR) antigen in phytohemagglutinin (PHA)-activated human peripheral blood T cells were examined. T cells were purified by a four-step procedure, which included depletion of glass-adherent cells, 53% Percoll gradient centrifugation, nylon wool column passage, and treatment with mouse monoclonal antibodies directed to human HLA-DR antigen and Leu M1 antigen plus complement. Purified T cells responded poorly to PHA but with the combination stimuli of PHA and recombinant human interleukin 2 (rIL-2), resting T cells proliferated as well as T cells cultured with 10% monocytes and PHA. But well proliferated T cells in the absence of monocytes expressed very poor DR antigen after 7 to 8 days of culture. DR expression of T cells was restored by the addition of 10% monocytes. Allogeneic monocytes also helped proliferative responses of PHA-activated T cells but did not help the expression of DR antigen. These results suggested that signals required for T cell proliferation (PHA and rIL-2) were not sufficient for DR expression in this system and further monocytes were essentially required in a HLA-restricted manner. In the next experiment, we examined the role of membrane molecules in monocytes for transmission of signals that induce activated T cells to express DR antigen. Autologous monocytes were fixed with 1% paraformaldehyde and added to T cells in the presence of PHA and rIL-2. Fixed monocytes could help DR antigen expression of PHA-activated T cells as well as viable monocytes. But when fixed monocytes were pretreated with anti-HLA-DR monoclonal antibody, they could not help DR expression of T cells any longer. These results suggested that for the expression of DR antigen, PHA-activated T cells had to first recognize self DR antigen expressed on the surface of monocytes before proliferation occurred.     

Monocyte-mediated augmentation of human natural killer cell activity: conditions, monocyte and effector cell characteristics

The characteristics of the effector cells and monocytes, and conditions required for the monocyte-mediated augmentation of human natural killer (NK) cell activity were investigated. Enriched null cell populations were further fractionated by Percoll centrifugation and used as effector cells. The LGL-enriched fraction was less susceptible than either the unfractionated cells or the other Percoll fractions to the monocyte augmentation when mixed with monocytes in the chromium-release assay and when precultured with monocytes for 12 hr, retrieved by carbonyl iron treatment, and tested for NK activity against K562. This differential susceptibility was reflected at the single cell level. The LGL-enriched Percoll fraction did not display the increase in target-binding cells with lytic activity that was exhibited by the other effector cell preparations after culture with monocytes. No differences in Leu-7 and Leu-11 phenotypes were detected between enriched null cells that had been cultured with and without monocytes for 12 hr. At the monocyte level, it was shown that pretreatment of the monocytes with LPS did not alter their NK-augmenting activity appreciably. Glutaraldehyde-fixed monocytes were not effective, and actinomycin D-treated monocytes were less effective than untreated or irradiated monocytes when mixed with enriched null cells in the assay. Actinomycin D-treated monocytes did not augment and possibly suppressed NK activity tested after 12-hr culture, and irradiated monocytes were less effective for augmenting NK activity than untreated cells. Monocyte-mediated augmentation could be detected when the medium used for null cell-monocyte coculture was supplemented with a) different lots of fetal bovine serum, b) human AB serum, c) autologous serum, or d) no serum. Polymyxin B and indomethacin did not alter the monocyte effect. Finally, the monocyte-mediated augmentation of human NK was not MHC restricted, since allogeneic combinations were also effective. These results suggest that 1) lymphocytes other than LGL participate in the monocyte-mediated augmentation of NK activity, 2) the augmentation is probably activational rather than maturational, 3) the monocytes must be viable to be effective when mixed with null cells during the assay, 4) de novo RNA and/or protein synthesis by the monocytes is required for the monocytes to induce augmented activity in null cells after 12-hr coculture, 5) prostaglandin synthesis and endotoxin are probably not involved in the augmentation, 6) the phenomenon is not MHC restricted, and 7) monocytes may express augmentative and suppressive activities concurrently.     

Interaction of C-reactive protein with lymphocytes and monocytes: complement-dependent adherence and phagocytosis.

The serum constituent C-reactive protein (CRP), which activates the classical complement (C) pathway when reacting with its substrates, was examined for its ability to mediate reactions of opsonic adherence and phagocytosis. Erythrocytes coated with C-polysaccharide (CPS) and reacted with CRP (E. CPS-CRP) failed to adhere to B cells and displayed only minimal adherence to monocytes. However, upon the addition of absorbed C or purified C components these cells were found to possess the cleavage products C4b and C3b, which in turn resulted in attachment of these cells to both human B lymphocytes and peripheral blood monocytes. E. CPS-CRP treated with C in the absence of antibody were readily phagocytosized by glass-adherent human monocytes. The phagocytosis of E. CPS-CRP-C was not only mediated by CRP but also required the presence of CRP on the surface of the red cells. The extent of ingestion was proportional to the amount of CRP on the red cell intermediate and was reduced by blocking monocyte receptors with aggregated human gamma-globulin (HGG) at concentrations which did not impair the uptake of other particles. The mediation by CRP of reactions of opsonic adherence and phagocytosis as outlined in these studies points to a significant role for CRP in reactions of host defense and inflammation.     

Motility, acrosome integrity, membrane integrity and oocyte cleavage rate of sperm separated by swim-up or Percoll gradient method from frozen-thawed buffalo semen

Frozen-thawed semen of five buffalo bulls was used to compare efficacy of swim-up and Percoll gradient methods for separating viable spermatozoa. Sperm separated by the two methods were also tested to differentiate buffalo bulls on the basis of in vitro fertilization (IVF) rates. Recovery of motile sperm (%), increase in membrane integrity (%) and acrosome integrity (%) were compared after two sperm separation methods in experiment I, and in vitro fertilization rate (cleavage rate and cleavage index) was compared in experiment II. Swim-up separated sperm showed a higher motility (P<0.05), while percent recovery of motile sperm was higher with Percoll separation (P<0.05). Membrane integrity (%) of sperm separated with swim-up was significantly higher (P<0.05) as compared to sperm separated with Percoll gradient. Swim-up separated sperm gave a higher cleavage rate and cleavage index (P<0.001). Sperm separated by swim-up showed significant difference among the bulls in cleavage rate and cleavage index (P<0.05), while the Percoll gradient method did not. It has been concluded that separation of sperm from frozen-thawed buffalo semen by swim-up method can be more expedient for IVF in buffalo.     

Chlamydia pneumoniae-infected monocytes exhibit increased adherence to human aortic endothelial cells

Interactions between monocytes and endothelial cells play an important role in the pathogenesis of atherosclerosis, and monocyte adhesion to arterial endothelium is one of the earliest events in atherogenesis. Work presented in this study examined human monocyte adherence to primary human aortic endothelial cells following monocyte infection with Chlamydia pneumoniae, an intracellular pathogen associated with atherosclerosis by a variety of sero-epidemiological, pathological and functional studies. Infected monocytes exhibited enhanced adhesion to aortic endothelial cells in a time- and dose-dependent manner. Pre-treatment of C. pneumoniae with heat did not effect the organism's capacity to enhance monocyte adhesion, suggesting that heat-stable chlamydial antigens such as chlamydial lipopolysaccharide (cLPS) mediated monocyte adherence. Indeed, treatment of monocytes with cLPS was sufficient to increase monocyte adherence to endothelial cells, and increased adherence of infected or cLPS-treated monocytes could be inhibited by the LPS antagonist lipid X. Moreover, C. pneumoniae-induced adherence could be inhibited by incubating monocytes with a mAb specific to the human beta 2-integrin chain, suggesting that enhanced adherence resulted from increased expression of these adhesion molecules. These data show that C. pneumoniae can enhance the capacity of monocytes to adhere to primary human aortic endothelial cells. The enhanced adherence exhibited by infected monocytes may increase monocyte residence time in vascular sites with reduced wall shear stress and promote entry of infected cells into lesion-prone locations.