Time course of plasma and pulmonary lymph endothelin-like immunoreactivity during sustained endotoxaemia in chronically instrumented sheep.

1. Endothelin, a novel vasoconstrictor 21-residue peptide isolated from the supernatant of cultured porcine endothelial cells, has been shown to be increased in plasma in a variety of cardiovascular disease states, including acute myocardial infarction, acute renal failure and essential hypertension. We determined the time course of plasma and pulmonary lymph endothelin-like immunoreactivity in relation to the progressive deterioration of cardiopulmonary function in an ovine septic shock model leading to multi-organ failure syndrome and death within 42 h of a continuous intravenous infusion of Escherichia coli endotoxin (40 ng min-1kg-1). 2. Plasma and pulmonary lymph endothelin-like immunoreactivity were measured by r.i.a. using a specific antiserum raised in rabbits against porcine endothelin-1. Endothelin-like immunoreactivity was further determined in lung tissue and the thoracic duct lymph of endotoxin-treated sheep by reversed-phase h.p.l.c. In control instrumented conscious sheep not infused with endotoxin, there were no significant changes in any of the measured cardiopulmonary and biochemical variables, with plasma and pulmonary lymph endothelin-like immunoreactivity remaining below the detection limit (less than 1 pg/tube) throughout the 72 h study period. 3. Conscious sheep receiving endotoxin showed a major hypotensive septic syndrome, including persistently decreased systemic blood pressure, systemic vascular resistance, stroke volume, left ventricular stroke work, associated with sustained pulmonary vasoconstriction and protein-rich pulmonary oedema (greater than five-fold increase in pulmonary lymph flow and protein clearance), and marked lactic acidosis, leading to the death of animals within 14-42 h despite institution of mechanical ventilation and adequate intravascular volume replacement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dysregulation of in vitro cytokine production by monocytes during sepsis.

The production by monocytes of interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF alpha) in intensive care unit (ICU) patients with sepsis syndrome (n = 23) or noninfectious shock (n = 6) is reported. Plasma cytokines, cell-associated cytokines within freshly isolated monocytes and LPS-induced in vitro cytokine production were assessed at admission and at regular intervals during ICU stay. TNF alpha and IL-6 were the most frequently detected circulating cytokines. Despite the fact that IL-1 alpha is the main cytokine found within monocytes upon in vitro activation of cells from healthy individuals, it was very rarely detected within freshly isolated monocytes from septic patients, and levels of cell-associated IL-1 beta were lower than those of TNF alpha. Cell-associated IL-1 beta and TNF alpha were not correlated with corresponding levels in plasma. Upon LPS stimulation, we observed a profound decrease of in vitro IL-1 alpha production by monocytes in all patients, and of IL-1 beta, IL-6, and TNF alpha in septic patients. This reduced LPS-induced production of cytokines was most pronounced in patients with gram-negative infections. Finally, monocytes from survival patients, but not from nonsurvival ones recovered their capacity to produce normal amounts of cytokines upon LPS stimulation. In conclusion, our data indicate an in vivo activation of circulating monocytes during sepsis as well as in noninfectious shock and suggest that complex regulatory mechanisms can downregulate the production of cytokines by monocytes during severe infections.     

Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies.

We used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. None of the monoclonal autoantibodies appeared to bind to a significant percentage of cells of relatively small cell size, either before or after culture. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Further experiments, including those using aggregated Ig to block antibody binding, strongly indicated that anti-histone antibody binding was not Fc receptor mediated. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations (0.25 micrograms/ml) of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.     

Differential processing of proenkephalin-A by human peripheral blood monocytes and T lymphocytes.

Human peripheral blood mononuclear cells are analyzed for preproenkephalin gene expression and peptide processing. Met-enkephalin immunoreactivity as detected with a specific antiserum is found in the cytoplasm of monocytes but not in T lymphocytes. Secretion of met-enkephalin was analyzed with an RIA that is specific for the met-enkephalin pentapeptide. Unfractionated PBMC spontaneously released 40 pg/ml met-enkephalin and this increased two- to fourfold after stimulation with PHA. Lower levels (less than 100 pg/ml) of met-enkephalin were detected in supernatants from purified T cells that were activated with PHA and IL-2. In contrast, stimulation of purified monocytes with LPS or PMA resulted in the release of up to 600 pg/ml of the processed peptide. To examine whether T cells can produce met-enkephalin precursor peptides, T cell conditioned media were treated with trypsin and carboxypeptidase-B, which is known to release met-enkephalin from the propeptide. This increased levels of met-enkephalin to 400 pg/ml, indicating that lymphocytes secrete the propeptide but do not process it to met-enkephalin. The 1.4-kb preproenkephalin mRNA is detected in activated blood mononuclear cells and in purified monocytes and T cells. To determine whether monocytes or lymphocytes express met-enkephalin in vivo, lymphoid tissues were analyzed by immunohistochemistry. In human spleen tissue, positive cells were found in the red pulp but not in the follicles, which is also consistent with met-enkephalin expression in monocytes. In summary, these results show that human peripheral blood mononuclear cells express preproenkephalin mRNA and that monocytes, but not T cells, process the propeptide to metenkephalin.     

人外周血Tie2-expressing monocytes的初步研究

目的:探讨健康人外周血中tyrosine kinase with Ig-like loops and epidermal growth factor homology domains-2(Tie2)-expressing monocytes(TEMs)的存在以及其所占的比例。方法:采用红细胞裂解法获得人外周血白细胞,Phycoerythrin(PE)标记抗人Cluster of Differentiation 14(CD14)抗体及Tie2抗体分别孵育所获取的白细胞,流式细胞仪检测CD14阳性细胞及Tie2阳性细胞的比例。结果:外周血白细胞中,CD14阳性率为2.04%,Tie2阳性率为0.71%。CD14阳性细胞中,Tie2阳性率为13.07%。结论:TEMs在健康人外周血白细胞中存在,并且主要分布于CD14阳性的单核细胞群。     

Detection of properdin mRNA in human peripheral blood monocytes and spleen.

Properdin serves a critical role in the alternative pathway of complement by stabilizing the C3bBb complex. Early studies to determine the properdin sequence relied on amino acid sequencing of enzymatically cleaved properdin and only yielded partial sequence data. Recently Nolan et al. reported a properdin mRNA sequence obtained from the U937 myelomonocytic cell line. We sought to detect properdin mRNA in two normal human tissues and to compare those sequences with that obtained from the U937 cell line. Cytoplasmic RNA harvested from human spleen and peripheral blood monocytes served as a template for first strand synthesis. The cDNA was then used as a template for polymerase chain reaction. A properdin message was detected in both spleen and peripheral blood monocytes but not in peripheral blood neutrophils. The sequence was nearly identical to that obtained from the U937 cell line. These experiments demonstrate that peripheral blood may be used as a ready source for properdin mRNA and will faster studies to define the defect in properdin-deficient patients.     

Increased angiotensin-converting enzyme in peripheral blood monocytes from patients with sarcoidosis.

Angiotensin-converting enzyme (ACE) activity was measured in isolated peripheral blood monocytes and culture medium from 28 patients with sarcoidosis and compared with values obtained from monocytes of 25 normal control subjects. ACE activity was determined by radioimmunoassay of angiotensin II produced from angiotensin I. While there was no measurable ACE activity in monocytes or culture medium from normal controls under the conditions of our study, monocytes from patients with sarcoidosis all showed activity both in cells and culture medium. The mean ACE activity of monocytes from patients with sarcoidosis was 2.0 pg angiotensin II formed/min per 10(5) cells, and that released into medium over a 24-h interval was 30.4 pg angiotensin II/min per 10(5) cells. The monocyte ACE from patients with sarcoidosis was activated by chloride ions and inhibited by EDTA, captopril, and rabbit antiserum to purified human plasma ACE, indicating that enzymatic activity was effected specifically by ACE. Thus, our studies show a significant elevation and release of ACE by peripheral blood monocytes of patients with sarcoidosis under conditions where monocytes of normal control subjects do not demonstrate ACE activity.     

Plasma fibronectin enhances phagocytosis of opsonized particles by human peripheral blood monocytes

We have investigated the effect of plasma fibronectin (Fn) on binding and phagocytosis of sheep erythrocytes (E) by human peripheral blood monocytes. Unopsonized E were not phagocytosed in the absence or presence of Fn, but Fn enhanced the phagocytosis of E bearing IgG. Sheep erythrocytes sensitized with IgM and C3b were ingested only when monocytes were exposed to Fn. The Fn enhancement of phagocytosis occurred for both fluid-phase and glass-adherent monocytes. Experiments in which Fn was washed out before mixing monocytes with opsonized E demonstrated that the Fn effect occurred because of interaction with the monocytes and not the opsonized particles. Chromatography of the Fn on Biogel A 1.5m showed that the phagocytosis-enhancing activity exactly co-chromatographed with the Fn protein. Fn did not increase the number of monocyte membrane receptors for the Fc fragment of monomeric IgG. We conclude that Fn enhances monocyte phagocytosis, not by binding to particles as a conventional opsonin, but by stimulating monocytes to ingest already opsonized particles more avidly.     

Differential sensitivity of peripheral blood granulocytes, monocytes, and lymphocytes to endotoxin-induced apoptosis in patients who have sustained Salmonella infection

The differential sensitivity of peripheral blood granulocytes, monocytes, and lymphocytes to endotoxin-induced apoptosis was studied in convalescents with Salmonella infection. The number of early (Tunel+PI-) and late (Tunel+PI+) apoptotic cells and the rate of apoptosis (the relative content of DNA apoptotic fragments in the solitary cell) were determined on a FACSCalibur cytofluorometer, by applying a package of the CellQuest programs (Becton Dickinson). A higher threshold of sensitivity of granulocytes, monocytes, and lymphocytes to the apoptotic effect of low, suboptimal doses (100 ng), as well as suppression of early stages of apoptosis by the optimal doses (1000 ng) of S. enteritidis lypopolysaccharides (LPS) were observed in patients who had sustained salmonellosis at a stage of reconvalescence. The hierarchy of LPS-induced apoptosis (granulocyte--monocyte--lymphocyte), observed in healthy donors, was also retained in the group of patients receiving the suboptimal doses of LPS, which seems to be associated with the repeated action of toxin on sensitized cell populations.     

Immunological studies on Crohn's disease. VIII. Diminished phagocytic activity in peripheral blood monocytes

We have been examining superoxide production activity in patients with Crohn's disease and we noted an increase in this activity in peripheral whole blood and monocytes, determined by a luminol-dependent chemiluminescence. However, superoxide production and phagocytosis are different phenomena. To search for the "real" phagocytic capacity, we examined the phagocytic activity of peripheral blood monocytes and neutrophils in 20 Japanese patients with Crohn's disease, using automated laser flow cytometry. In contrast to the results obtained with the chemiluminescence assay, we found a decrease in the "real" phagocytic function in monocytes, yet there was no difference in the phagocytic ability in neutrophils. The phagocytic activity in patients with an active form of the disease was lower than that in patients with inactive form of the disease. These results provide pertinent information on the role of monocytes in Crohn's disease. The depressed phagocytic activity in monocytes from these patients may play some role in the pathogenesis of this disease.     

Interleukin 1 alpha production by peripheral blood monocytes from patients with chronic liver disease and effect of sera on interleukin 1 alpha production

We investigated the role of interleukin 1 alpha (IL 1 alpha) in the pathogenesis of chronic liver disease. IL 1 alpha production by peripheral blood monocytes was measured with a specific, sensitive double-antibody radioimmunoassay. When monocytes were cultured for two days with bacterial lipopolysaccharide (LPS), IL 1 alpha production in asymptomatic hepatitis B virus carrier (ASC) and patients with chronic active hepatitis (CAH) was equivalent to that of controls (168 +/- 31 U/ml, mena +/- SD), while IL 1 alpha levels generated by monocytes from liver cirrhosis (LC) (117 +/- 45 U/ml, p less than 0.01) were significantly lower than controls. When normal monocytes were cultured together with LPS and IFN gamma, mena IL 1 alpha production was 297 +/- 56 U/ml. IL 1 alpha production in ASC did not differ from controls. On the other hand, IL 1 alpha production in patients with CAH (241 +/- 58 U/ml, p less than 0.05) and LC (189 +/- 70 U/ml, p less than 0.01) were significantly diminished in comparison with controls although there was considerable overlap. Serial study demonstrated that IL 1 alpha production rose significantly during acute deterioration of illness with marked rise in serum alanine aminotransferase. The addition of sera to normal monocytes cultures resulted in significantly enhanced suppression (p less than 0.05) for IL 1 alpha production in comparison with that of control sera. These findings indicate that decreased monocyte function and serum inhibitor(s) for IL 1 alpha production could contribute to the pathogenesis of chronic liver disease.     

Superoxide production by phagocytic leukocytes

Mononuclear phagocytic leukocytes, as well as polymorphonuclear leukocytes, produce and release superoxide at rest, and this is stimulated by phagocytosis. Of the mouse monocytic cells studied, alveolar macrophages released the largest amounts of superoxide during phagocytosis, followed by normal peritoneal macrophages. Casein- elicited and "activated" macrophages released smaller quantities. In the guinea pig, polymorphonuclear leukocytes and casein-elicited macrophages were shown to release superoxide during phagocytosis whereas alveolar macrophages did not. Superoxide release accounted for only a small fraction of the respiratory burst of phagocytosis in all but the normal mouse peritoneal macrophage, the guinea pig polymorphonuclear leukocyte, and probably the mouse alveolar macrophage. There are obviously considerable species differences in O2- release by various leukocytes that might reflect both the production and/or destruction (e.g. by dismutase) of that substance.     

Prostaglandin E production by human blood monocytes and mouse peritoneal macrophages

Purified populations of both human peripheral blood monocytes and murine peritoneal macrophages synthesize and release Prostaglandin E in vitro. In contrast, prostaglandin E was detected in neither the supernate fluids from cultures of highly enriched human lymphocytes and granulocytes, nor in nonadherent murine peritoneal cells. Macrophage prostaglandin E production was markedly enhanced by endotoxin, and completely suppressed by indomethacin. All neoplastic monocyte-macrophage cell lines examined elaborated prostaglandin E in vitro, either constitutively or after induction with endotoxin. In contrast, prostaglandin E production could not be detected from either a T- or B-cell lymphoma, whether or not they were treated with endotoxin. These findings thus indicate that the blood monocyte and tissue macrophage represent an important source of prostaglandin E, a function shared by both normal and neoplastic mononuclear phagocytes.     

Cocaine-mediated suppression of superoxide production by human peripheral blood mononuclear cells.

Cocaine, like opiates, modulates a variety of immune functions. In the present study, we investigated the effect of cocaine on superoxide anion (O2-) production, an index of a microbicidal activity, by cultured human peripheral blood mononuclear cells. Release of O2- was measured by superoxide dismutase-inhibitable reduction of ferricytochrome C in response to phorbol myristate acetate. Peripheral blood mononuclear cells cultured in the presence of cocaine (1 microM) for 48 hr released less (P less than .05) O2- than did nontreated control cells (95.1 +/- 10.2 vs. 57.9 +/- 6.6 nmol/10(7) cells/60 min, respectively). This suppressive effect was dose-dependent. Antibodies to transforming growth factor-beta, a cytokine inhibitory of monocyte O2- production, abrogated (P less than .01) cocaine-mediated suppression, suggesting that transforming growth factor-beta is involved in the suppression. Also, naloxone blocked (P less than .01) the suppressive effects of both cocaine and transforming growth factor-beta on O2- production, suggesting that the suppressive mechanism is naloxone-sensitive.