Prostaglandin E2 production by rat peritoneal macrophages: role of cellular and humoral factors in vivo in transfusion-associated immunosuppression

Abstract The transfusion of blood is associated with long-term immunosuppression, which has been postulated to influence immunosurveillance and cancer cell killing. The mononuclear phagocyte synthesises large quantities of PGE₂, and PGE₂ has been shown to inhibit the activity of a range of immunocompetent cell types. The role of mononuclear phagocyte PGE₂ synthesis in transfusion-associated immunosuppression, and the elements of transfused blood which control this immunosuppression, were investigated using a transfused rat model. A significant increase in macrophage PGE₂ synthesis was detected 7 days after transfusion with blood and serum. The storage of blood for 24 h increased the stimulatory activity of transfused blood. The effects of storage and serum on macrophage PGE₂ synthesis were greater than effects due to genetic differences between blood donor and recipient, and the serum effects indicated that a major factor activating PGE₂-mediated immunosuppression in transfused subjects may be humoral in nature.     

Macrophage prostaglandin E2 and oxidative responses to endotoxin during immunosuppression associated with anaesthesia and transfusion

The widespread use of blood transfusion in major surgical procedures has led to concern about the immunosuppressive effect of transfusion on patients with underlying malignancy. Transfusion may also suppress the host response to infection. The cellular mechanisms of transfusion-associated immunosuppression may involve macrophage prostaglandin E₂ (PGE₂) in modulating the host response to cancer and infection. We previously observed that the transfusion of blood increased PGE₂ production by unstimulated macrophages. To investigate this PGE₂ associated immunosuppression, we studied the effect of transfusion of rats using a physiological stimulus of macrophage PGE₂ production, bacterial endotoxin. In the same macrophages, we analysed intracellular oxidative activity. Both allogeneic and syngeneic blood transfusion were associated with increased PGE₂ release by macrophages. This stimulation of PGE₂ increased with duration of storage of blood. A similar effect of serum indicated that a humoral factor was involved. Endotoxin (50 ng/ml–500 μg/ml) stimulated PGE₂ production in all transfused subjects. The lowest endotoxin concentration gave proportionately the greatest stimulation. Oxidative activity was down-regulated in macrophages of transfused rats, supporting an immunosuppressive role of PGE₂ within the macrophage. An effect of surgery on the oxidative response was also detected.     

Cytochemical reactions of human hematopoietic cells in liquid culture.

Growth of human bone marrow in liquid suspension cultures has been used to study normal hematopoietic cell differentiation and abnormalities in blood diseases. A variety of cytochemical stains were applied to human marrow cells cultured in vitro for up to 14 days. AS-D- CHLOROACETATE ESTERASE AND ALPHA-NAPHYHYL BUTYrate esterase were most useful in distinguishing different cell lines in culture. Peroxidase activity disappeared with mononuclear phagocyte morphogenesis and diminished with culture in intermediate and mature granulocytes. Acid phosphatase activity and methyl greed pyronin staining intensity increased with macrophage maturation.     

Collection, storage and transfusion of blood stem cells for the treatment of hemopoietic failure.

Migration of hemopoietic stem cells via the blood to sites of stem cell need is a principle that becomes established during the embryonic development of hemopoiesis and can be observed in the adult whenever bone marrow transplantations are being performed. The regular presence of stem cells in the peripheral blood lends itself to the study of their collection, storage, and use for transfusion purposes in cases of bone marrow failure. Both in dog and in man, granulocyte-macrophage progenitor cells (CFU-C) can be collected by leukapheresis from the blood in large quantities, particularly if the yield is increased by the administration of mobilizing agents such as dextran sulfate, and appear to be an indicator for the presence of stem cells. For collection and storage, a closed plastic bag system has been developed that allows the safe handling of the cells. The loss of CFU-C from freezing and thawing with DMSO as a cryoprotective agent is only 10%-20%. If frozen and thawed mononuclear leukocytes are transfused into 1200 rad whole-body X-irradiated autologous or allogeneic recipient dogs, a hemopoietic take is observed when 0.2 X 10(5) CFU-C are present among the mononuclear leukocytes (MNC). Graft-versus-host disease can be avoided in the allogeneic situation when a purified CFU-C rich cell fraction is being transfused. In man collection and storage of MNC including CFU-C is feasible and may eventually become a therapeutic tool.     

Tartrate-resistant acid phosphatase in human alveolar macrophages

Tartrate-resistant acid phosphatase has been extracted from human alveolar macrophages, in which its specific activity is 10-fold that in whole lung. The apparent identity of the alveolar macrophage isoenzyme with that associated with osteoclasts suggests that both types of cell belong to the mononuclear phagocyte system. Within this system, expression of tartrate-resistant acid phosphatase appears to accompany certain kinds of differentiation.     

Red cell storage

Blood component storage allows the donor and recipient to be separated in time and space. This separation converts transfusion from a desperate clinical act into a planned, orderly healthcare logistic activity with concomitant increases in both blood product availability and safety. However, storage has the potential to reduce the efficacy of transfused blood components by reducing their flow, functional capacity, and survival. Storage time also allows the accumulation of leaked potassium from red cells and the growth of contaminating bacteria. Many different aspects of the red cell storage lesion have been described, including changes in metabolism, shape, and rheology changes, loss of membrane carbohydrates, lipids and proteins, and alterations in secretion, oxygen delivery, and adhesion. What has been harder to show is that these known changes have significant clinical effects. Therefore, regulatory decisions about product storage have been conservative, and largely based on historic patterns of use. The increasing power of proteomics and metabolomics offers the potential of deeper understanding of blood function and storage and of better clinical products in the future.     

Prostaglandins regulate melanoma-induced cytokine production in macrophages

Tumor-secreted products can affect macrophage cytokine expression and in that way alter the immune response. Prostaglandins (PGs) are found in the tumor microenvironment and have been associated with local and regional immunosuppression. We investigated whether tumor-secreted factors could induce PG synthesis in macrophages and whether these PGs could alter macrophage production of immunoregulatory cytokines. In both murine and human models, melanoma conditioned medium (MCM) induced macrophage production of PGE(2), IL-6, and TNF-alpha. PGE(2) production increased over 24 h and was accompanied by an increase in cyclooxygenase-2 (COX-2) expression, while COX-1 expression remained unchanged. In the presence of 10 microM NS398, a selective COX-2 inhibitor, MCM-stimulated PGE(2) synthesis was almost completely suppressed, while production of IL-6 and TNF-alpha proteins and mRNA also was partially abrogated. In the murine model, 200 microM NS398 resulted in more significant inhibition of cytokine protein and mRNA production. Although MCM induced NFkappaB and NF-IL-6 activation, neither dose of NS398 altered this effect. We conclude that melanoma-secreted products stimulate COX-2 expression and PGE(2) synthesis in macrophages and that inhibition of COX-2-derived PG synthesis results in partial abrogation of macrophage cytokine production.     

Decreased sensitivity of atopic mononuclear cells to prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2)

The effect of PGE2 and PGD2 on several lymphocyte functions in vitro was evaluated in nonatopic and atopic subjects. Both PGE2 and PGD2 inhibited phytohemagglutinin-induced protein synthesis ([3H] leucine uptake) by nonatopic mononuclear cells and T cells in a dose-dependent manner (10(-6) to 10(-12) M). Protein synthesis by atopic mononuclear cells was not significantly suppressed by the above concentration of PGE2. Although PGD2 effectively suppressed protein synthesis by atopic mononuclear cells and T cells at 10(-6) M, lower concentrations were ineffective. Kinetic studies revealed significant differences in the suppressive effects of PGE2 and PGD2 on atopic and nonatopic mononuclear cells at 24 and 48 h, but not at 72 or 96 hr. Protein synthesis by T helper-enriched populations (suppressor cell depletion by anti-Leu-2b + complement) obtained from nonatopics was significantly reduced by PGE2 and PGD2, suggesting that these mediators may be directly inhibiting the responding population. By contrast, protein synthesis by T suppressor-enriched populations (helper cell depletion by OKT4 + complement) obtained from nonatopics was enhanced by PGE2 and PGD2, suggesting that the PG were activating these cells. Atopic T helper and T suppressor cells exhibited decreased responsiveness to PGE2 and PGD2 compared with nonatopic cells. PGE2 and PGD2 inhibited the phytohemagglutinin-stimulated proliferative response ([3H]thymidine uptake) by both atopic and nonatopic mononuclear cells in a dose-dependent manner and to the same extent. However, although PGE2 and PGD2 generated functional suppressor activity (when using a coculture technique) in nonatopic mononuclear cells, these mediators failed to activate atopic suppressor cells. These results suggest that reduced responses by atopic T cells to signals provided by PGE2 and PGD2 are not solely restricted to suppressor cell function, and could indicate an impaired ability to regulate immune and/or inflammatory reactions.     

成份输血对胃癌术后血清IL-6、IL-10、COX-2、PGE_2含量的影响

目的：观察围手术期成份输血对胃癌患者炎症反应的影响。方法：随机选择胃癌病人分为3组：对照组,压积红细胞组,全血组。分别采用ELISA法和放免法检测患者术前及术后低1、3、7、14天血清中IL-6、IL-10、COX-2及PGE2含量。结果：手术后第一周是感染发生的主要时期,两周后,炎症反应基本消失,输血能升高患者血清中的促炎因子,降低抗炎因子IL-10的表达,但相对于输异体全血,输成份血能降低血清中COX-2、PGE2、IL-6的含量,升高血清IL-10的含量。结论：胃癌患者围手术期输血能促进机体的炎症反应,成份输血致炎效果弱于输异体全血。     

Some mechanisms involved in the prostaglandin E2 immunosuppressive effect in (CBA x C57BL)F1 mice in vivo

The cellular mechanisms involved in the immunosuppressive effect of prostaglandin E2 (PGE2) multiple injections into (CBA x C57Bl)F1 mice in vivo have been studied. PGE2 injection increases the induction of specific T-suppressors. In addition, there is a decrease in macrophage phagocytic activity and in the phagocytosis index, apparently mediated by Fc gamma receptors (Fc gamma R) and not by the macrophage complement receptor (C3R). The induction of antibody synthesis by using "immune" macrophages injected into a syngeneic recipient results in considerable decrease in the accumulation of antibody-forming cells if the macrophage donor has been pretreated with exogenous PGE2 in comparison with untreated controls. These cellular mechanisms are possibly one part of the diverse way in which PGE2 exerts an immunosuppressive effect in vivo and contributes to humoral immune response suppression.     

双歧杆菌对小鼠单核吞噬细胞功能的影响

双歧杆菌是革兰氏阳性无芽胞厌氧菌,是人和动物肠道的正常菌群之一。我们研究了注射双歧杆菌对小鼠单核吞噬细胞功能的影响。注射婴儿双歧杆菌和青春双歧杆菌后小鼠腹腔巨噬细胞酸性磷酸酶含量增加、吞噬试验的吞噬率及吞噬指数明显提高,表明双歧杆菌能增加巨噬细胞吞噬消化功能,以婴儿双歧杆菌为启动剂可从DBA/2小鼠体内诱生肿瘤坏死因子,提示双歧杆菌可调节单核吞噬细胞分泌细胞因子。因此双歧杆菌能激活单核吞噬细胞,促进机体的免疫学反应。推测定居于肠道的双歧杆菌可能是通过移位到体内器官、释放免疫活性成分被肠道中Peryer氏淋巴结群内的巨噬细胞吞噬,从而作用于机体单核吞噬细胞系统。这一推测尚需进一步研究证实。     

Effect of prostaglandin E2 on PMA-induced macrophage differentiation

Major trauma such as severe bums and extensive surgery could result in accelerated macrophage differentiation and hyperactivation causing an excessive release of proinflammatory cytokines and prostaglandin E2 (PGE2) with consequent severe impairment of immunologic reactivity. HL-60 cells stimulated with phorbol 12-myristate 13-acetate (PMA) have been used as a model to asses the PGE2 role in the macrophage differentiation observed after major trauma. Cell adhesion, matrix metalloproteinase-9 (MMP-9) and tumor necrosis factor-alpha (TNF-alpha) production were measured after 24 h of PMA treatment in the presence of PGE2 (1 nM - 1 microM). PGE2 increased both the PMA-induced cell adhesion and MMP-9 production via EP2/EP4 receptors while it had no effect on the induced TNF-alpha release. The cAMP/PKA pathway, usually linked to EP2/EP4 activation, was not involved in the phenomenon, suggesting that an alternative signalling pathway could be linked to a PKC-activated enzyme. In fact PGE2 activity was partially inhibited by Wortmannin, a phosphoinositide-3 kinase (PI-3K) inhibitor indicating that PGE2 act as a co-factor able to increase macrophage differentiation in vitro via a PI-3K dependent pathway that could be also involved in the immunosuppression observed in the aftermath of trauma.     

Neutrophils recruited to the site of Mycobacterium bovis BCG infection undergo apoptosis and modulate lipid body biogenesis and prostaglandin E production by macrophages

Neutrophil influx to sites of mycobacterial infections is one of the first events of tuberculosis pathogenesis. However, the role of early neutrophil recruitment in mycobacterial infection is not completely understood. We investigated the rate of neutrophil apoptosis and the role of macrophage uptake of apoptotic neutrophils in a pleural tuberculosis model induced by BCG. Recruited neutrophils were shown to phagocyte BCG and a large number of neutrophils undergo apoptosis within 24 h. Notably, the great majority of apoptotic neutrophils were infected by BCG. Increased lipid body (lipid droplets) formation, accompanied by prostaglandin E(2) (PGE(2)) and TGF-beta1 synthesis, occurred in parallel to macrophage uptake of apoptotic cells. Lipid body and PGE(2) formation was observed after macrophage exposure to apoptotic, but not necrotic or live neutrophils. Blockage of BCG-induced lipid body formation significantly inhibited PGE(2) synthesis. Pre-treatment with the pan-caspase inhibitor zVAD inhibited BCG-induced neutrophil apoptosis and lipid body formation, indicating a role for apoptotic neutrophils in macrophage lipid body biogenesis in infected mice. In conclusion, BCG infection induced activation and apoptosis of infected neutrophils at the inflammatory site. The uptake of apoptotic neutrophils by macrophages leads to TGF-beta1 generation and PGE(2)-derived lipid body formation, and may have modulator roles in mycobacterial pathogenesis.     

Proteomics and transfusion medicine: future perspectives

Limited number of important discoveries have greatly contributed to the progresses achieved in the blood transfusion; ABO histo-blood groups, citrate as anticoagulant, fractionation of plasma proteins, plastic bags and apheresis machines. Three major types of blood products are transfused to patients: red cell concentrates, platelet concentrates and fresh frozen plasma. Several parameters of these products change during storage process and they have been well studied over the years. However, several aspects have completely been ignored; in particular those related to peptide and protein changes. This review presents what has been done using proteomic tools and the potentials of proteomics for transfusion medicine.     

Mechanism of viral infection of macrophages by an agent of hemorrhagic fever with renal syndrome: ultrastructural aspects

Monocytes/macrophages are one of the first cells subjected to the infectious effect of viruses. The present paper analyses for the first time the ultrastructural changes in macrophages caused by an agent of hemorrhagic fever with renal syndrome (HERS)--hantavirus (HV). After a local fusion with the host cell plasmalemma and its adsorption on the cell surface, the HV penetrates through the macrophage membrane. This process occurred without destruction of cell plasmalemma. HV viral particles were observe within the macrophage cytoplasm mostly on the smooth granular endoplasmic reticulum vesicles. Viroplasts were defined in macrophages after a 2 h incubation, with synthesis of viral nucleoproteins and primary covers being observed on the surface of viroplasts. Viral particles left macrophages in the process of budding on the phagocyte surface. Thereby HV, similar to other enveloped viruses, realizes entrance and egress from the target cell without damaging its plasmalemma. This accounts for the viral ability to reproduce in macrophages for a long time without any cytopathological effect. Consequently, in the absence of obvious destruction changes, mononuclear phagocytes can serve as a long-term storage of viruses, and thus being involved in HV dissemination during HERS.     

Opioid-mediated suppression of cultured peripheral blood mononuclear cell respiratory burst activity

Opiate addiction and stress have been associated with altered immune responses. In this study, we evaluated the influence of morphine and the stress responsive opioid peptide beta-endorphin (beta-END) on O-2 and H2O2 production by cultured human peripheral blood mononuclear cells. Exposure of these cells during 48 hr of culture to morphine and beta-END at pharmacologically (10(-8) M) and physiologically (10(-12) M) relevant concentrations, respectively, markedly suppressed peripheral blood mononuclear cell O-2 and H2O2 release in response to the respiratory burst stimuli opsonized zymosan and phorbol myristate acetate. Both opioids also induced a minimal, but statistically significant, increase in resting O-2 and H2O2 generation. The modulatory effects of morphine and beta-END on peripheral blood mononuclear cell oxygen metabolism appeared to involve a classical opioid receptor, because opioid activity was blocked by naloxone and was not observed with N-acetylated-beta-END. Using purified lymphocyte and monocyte preparations, we determined that although opioids directly increase monocyte-resting oxygen metabolism, lymphocytes are the primary target cell in opioid-mediated suppression of monocyte respiratory burst activity. The release of a suppressive product from opioid-triggered lymphocytes was inhibited by cyclosporine. Based on the results of this study, we propose that opioid-mediated suppression of mononuclear phagocyte respiratory burst activity is another factor to be considered in the immunodeficiency of opiate addiction and stress.     

Regulation of macrophage collagenase, prostaglandin, and fibroblast-activating-factor production by anti-inflammatory agents: different regulatory mechanisms for tissue injury and repair

Activation of macrophages results in the production of tissue destructive mediators and enzymes including prostaglandins (PGE) and collagenase. In addition, activated macrophages also generate mediators which enhance connective tissue formation through their effects on fibroblast growth. To determine whether the pro-inflammatory mediators and the mediator(s) involved in tissue repair are under the same regulatory control, guinea pig macrophage cultures were treated with various pharmacologic agents and their supernatants monitored for biologic activity. The nonsteroidal anti-inflammatory agent, indomethacin, and the glucocorticoid, dexamethasone, at pharmacologic concentrations inhibited not only prostaglandin synthesis (greater than 90%) but also the production of collagenase (greater than 90%). Colchicine, a microtubule disruptive agent, but not the inactive form, lumicolchicine, markedly diminished the production of collagenase independently of prostaglandin synthesis. In contrast to the inhibitory effects of these anti-inflammatory agents on PGE and collagenase production, indomethacin did not inhibit the production of macrophage-derived fibroblast-activating factor (FAF). Furthermore, dexamethasone at pharmacologic doses did not inhibit FAF production. Colchicine not only did not inhibit FAF, but frequently enhanced the appearance of FAF In the macrophage cultures. Thus, it appears that regulation of the production of PGE and collagenase is different than the regulation of FAF synthesis and therefore the production of these mediators can be differentially modulated. Such a dissociation may provide a basis for mononuclear cell-mediated fibroblast growth and tissue repair to occur independently of the release of PGE2 and collagenase and even following anti-inflammatory drug therapy.     

Analysis of the shelf life and transfusion life of whole blood and erythrocyte concentrates

The shelf life of 60,000 units of whole blood and red cell concentrates in the blood center were analysed before delivering to hospitals as well as the transfusion age of 18,000 units in relation to the stock-size and the outdated units. The mean transfusion age of whole blood units and red blood cell concentrates amounted to 11 to 12 days and 16 to 17 days, respectively. The shelf life of blood transfused within 24 hours after collection was 1.6% and 3.8% transfused within 48 hours according to the total number of whole blood and red blood cell concentrates. Washed red cell concentrates were prepared from buffy coat-free resuspended red cell concentrates on an average at the 7th day of storage. A high stock-size of about 1,000 units rather than of 600 units in the blood center increased the shelf life and also the number of outdated units from less than 0.5% up to more than 3%.     

Stimulation of prostaglandin E2 synthesis in cloned osteoblastic cells of mouse (MC3T3-E1) by epidermal growth factor

Prostaglandin (PG) E2, known as a bone-resorption factor, was released as a predominant arachidonate metabolite in the culture medium of an osteoblastic cell line cloned from mouse calvaria (MC3T3-E1). Epidermal growth factor (EGF) (10 ng/ml) prominently enhanced endogenous PGE2 synthesis, requiring the simultaneous presence of unidentified factor(s) contained in bovine serum. PGE2 synthesis increased after a lag phase for 1-2 h and reached a maximum level at about 3 h after EGF addition. EGF-stimulated PGE2 synthesis was almost completely blocked by 10 microM cycloheximide or 1 microM actinomycin D. Furthermore, when the cells were pretreated with EGF, the microsomes exhibited an increased activity of fatty acid cyclooxygenase (arachidonic acid----PGH2), whereas the activity of PGE synthase (PGH2----PGE2) remained unchanged. These results suggested an EGF-mediated induction of cyclooxygenase. Following increased PGE2 synthesis, DNA synthesis increased and alkaline phosphatase activity decreased in a slower response to EGF. PGE2 (above 0.1 microM) added to the cells could replace EGF. However, such effects of EGF on the osteoblasts could not be attributed totally to an autocrine function of PGE2 produced by stimulation with EGF because these effects of EGF were not abolished by indomethacin, which blocked the PGE2 synthesis.     

Effects of prostaglandins on LDL receptor activity and cholesterol synthesis in freshly isolated human mononuclear leukocytes

The effects of prostaglandin (PG) E1, PGE2, the stable prostacyclin analogue Iloprost, and PGF2 alpha on low density lipoprotein (LDL) receptor activity and cholesterol synthesis were investigated in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 45 hr in a lipid-free medium resulted in an increase in the rate of cholesterol synthesis from [14C]acetate and the high affinity accumulation and degradation of 125I-labeled LDL. Addition of PGE1 in increasing concentrations to the incubation medium inhibited cholesterol synthesis and the specific accumulation and degradation of 125I-labeled LDL; at a concentration of 10 microM, the inhibitions were 61%, 70%, and 67%, respectively, after an incubation of 20 hr. The effects of PGE2 and Iloprost were similar. The action of the prostaglandins on LDL receptor activity appeared to be mediated by a decrease in the number of LDL receptors and not by a change in the binding affinity. The prostaglandins yielded sigmoidal log concentration-effect curves. In contrast, PGF2 alpha had no influence on cholesterol synthesis or LDL receptor activity up to a concentration of 10 microM. PGE1, PGE2, and Iloprost, but not PGF2 alpha, led to an increase in the concentration of intracellular cyclic AMP. Dibutyryl cyclic AMP mimicked the effects of the E-prostaglandins and Iloprost on the LDL receptor activity. The results suggest that PGE1, PGE2, and prostacyclin affect LDL receptor activity and cholesterol synthesis and, therefore, may play a role in the regulation of cholesterol homeostasis and in the development of atherosclerosis.