Procoagulant Activity Production Research Articles

Effects of the second-generation synthetic lipid A analogue E5564 on responses to endotoxin equine whole blood and monocytes

To evaluate proinflammatory effects of the second-generation synthetic lipid A analogue E5564 on equine whole blood and isolated monocytes and to determine the ability of E5564 to prevent LPS (lipopolysaccharide)-induced procoagulant activity (PCA); tumor necrosis factor (TNF)-alpha production; and mRNA expression of TNF-alpha, interleukin (IL)-1beta, IL-6, and IL-10 by equine monocytes. Venous blood samples obtained from 19 healthy horses. Whole blood and monocytes were incubated with Escherichia coli O111:B4 LPS, E5564, or E5564 plus E coli O111:B4 LPS. Whole blood and cell supernatants were assayed for TNF-alpha, and cell lysates were assayed to determine PCA. Expression of mRNA for TNF-alpha, IL-1beta, IL-6, and IL-10 by monocytes was determined by use of real-time quantitative PCR assay. Minimal proinflammatory effects were detected in whole blood and monocytes. In addition, E5564 inhibited LPS-induced PCA and TNF-alpha production in a concentration-dependent manner. Furthermore, E5564 significantly inhibited LPS-induced mRNA expression of TNF-alpha, IL-1beta, and IL-10 and decreased LPS-induced expression of IL-6. The second-generation synthetic lipid A analogue E5564 lacked agonist activity in equine whole blood and monocytes and was a potent antagonist of enteric LPS. Therefore, E5564 appeared to be the first lipid A analogue that has potential as an effective therapeutic agent in horses with endotoxemia.

Intracellular antioxidant activity is necessary to modulate the macrophage response to endotoxin.

The tissue-fixed macrophage (Mphi) is a key cell in the coordination of the excessive systemic immunoinflammatory response underlying the adult respiratory distress syndrome (ARDS). Macrophage-generated reactive oxygen intermediates (ROIs) are involved in both tissue destruction via lipid peroxidation and in the activation of these inflammatory cells. It is unclear whether oxidant-induced activation involves an extracellular effect and membrane destabilization or occurs through intracellular alteration of the redox state and direct involvement as second messengers. In this study, we compare the differential effects of known intracellular vs. extracellular antioxidants on the Mphi response to endotoxin. Rabbit alveolar Mphi were obtained by bronchoalveolar lavage and exposed to either the extracellular antioxidants [vitamin C (VC) (10-1000 microM), Trolox (100-1000 microM, superoxide dismutase (SOD) (10-500 microM))] or the intracellular antioxidants [N-acetylcysteine (NAC) (0.1-10 mM) or butylated hydroxyanisole (BHA) (10-200 microM)] for 1 h. Cells were subsequently stimulated with lipopolysaccharide at 10 ng/mL. After 18 h, supernatants were analyzed for tumor necrosis factor (TNF) and F2 isoprostane (F2ISP) production and cellular monolayers for procoagulant activity (PCA). A dose response inhibition of both TNF and PCA production was demonstrated after both NAC and BHA pretreatment but not with VC, Trolox, or SOD. In addition, northern blots revealed inhibition of TNF mRNA production by both NAC and BHA. F2ISP, a marker of membrane lipid peroxidation, was inhibited by BHA and Trolox but not NAC, VC, or SOD. In conclusion, antioxidants that are incorporated intracellularly are expected to be beneficial in the treatment of excessive inflammatory responses through the interruption of redox dependent signal transduction pathways and subsequent modulation of the Mphi proinflammatory response.

The macrophage response to endotoxin requires platelet activating factor.

Platelet activating factor (PAF) is a key proinflammatory mediator of septic shock and is metabolized by PAF-acetylhydrolase (PAF-AH). Low circulating levels of PAF-AH have been associated with the development of autodestructive excessive inflammatory responses such as post-injury multiple organ failure, and recombinant PAF-AH is being studied for the prevention of acute respiratory distress syndrome (ARDS). However, the potential role of PAF as an autocrine mediator of macrophage activation is unclear. We wanted to examine the role of PAF in the endotoxin- (LPS) induced macrophage response using PAF-AH. Rabbit alveolar macrophages were stimulated with LPS (10 ng/mL) with or without PAF-AH (0.1-10 microg/mL). Supernatants were collected to measure the production of tumor necrosis factor (TNF), interleukin 8 (Il-8), and prostaglandin E2 (PGE2). Cell monolayers were assessed for procoagulant activity (PCA). TNF mRNA production was determined by Northern blot and RNA stability was assessed. Evaluation of intracellular signaling pathways for LPS included western blots for phosphorylated p38 and ERK kinases and gel shift for nuclear factor-kappaB. There was a dose-response inhibition of TNF, PCA, Il-8, and PGE2 production following pretreatment with PAF-AH. Time course studies revealed effective inhibition of TNF production with administration of PAF-AH up to 2 h after LPS challenge. TNF mRNA production was inhibited, while mRNA stability was not affected. There was no effect on the phosphorylation of p38 or ERK 1 kinases; however, the nuclear translocation of NF-kappaB was inhibited. Macrophage cytokine production in response to endotoxin is PAF dependent. This effect involves the inhibition of TNF gene transcription and concomitant inhibition of NF-kappaB.

Resistance of naive mice to murine hepatitis virus strain 3 requires development of a Th1, but not a Th2, response, whereas pre-existing antibody partially protects against primary infection.

Murine hepatitis virus strain 3 (MHV-3) produces a host-strain-dependent spectrum of disease. The development of liver necrosis has been shown to be related to production of a unique macrophage procoagulant activity (PCA), encoded by the gene fgl-2, in susceptible mice. These studies were designed to examine the influence of Th1/Th2 cells on resistance/susceptibility and production of macrophage procoagulant activity (PCA) in resistant (A/J) and susceptible (Balb/cJ) strains of mice following infection with MHV-3. Immunization of A/J mice with MHV-3 induced a Th1 cellular immune response and one Th1 cell line (3F9.1) protected susceptible mice and inhibited production of PCA by macrophages both in vitro and in vivo. In contrast, immunization of Balb/cJ mice with an attenuated variant of MHV-3 derived from passaging MHV-3 in YAC-1 cells resulted in a Th2 response. Transfer of spleen cells and T cell lines from immunized Balb/cJ mice failed to protect naive susceptible syngeneic mice from infection with MHV-3 and augmented production of IL-1 beta, TNF-alpha and PCA by macrophages to MHV-3 in vitro. Serum from immunized Balb/cJ mice contained high titered neutralizing antibody which protected naive Balb/cJ animals from lethal primary MHV-3 infection. These results demonstrate that susceptible Balb/cJ mice generate a Th2 response following MHV-3 infection and that these Th2 cells neither inhibit MHV-3-induced macrophage PCA production nor protect naive mice from MHV-3 infection. The results suggest that antibody protects against primary infection, but could not eradicate ongoing infection. Ribavirin, a synthetic guanosine analogue prolonged survival to MHV-3 infection, inhibited production and transcription of the macrophage pro-inflammatory cytokines IL-1 beta and TNF-alpha and Th2 cytokines while preserving Th1 cytokine production. Thus, this data defines the differential role of Th1/Th2 lymphocytes in primary and secondary MHV-3 infection and further defines the importance of macrophage inflammatory mediators in the pathogenesis of MHV-3 infection.

Induction of procoagulant activity in virus infected bovine alveolar macrophages and the effect of lipopolysaccharide

Three viruses known to be associated with the bovine respiratory disease complex were evaluated in vitro for potential impact upon the procoagulant activity (PCA) of bovine alveolar macrophages (bAM). Cultures of bAM were inoculated with bovine parainfluenza virus Type 3 (PI-3), cytopathic bovine viral diarrhea virus (cpBVDV), non-cytopathic BVDV (ncpBVDV), or bovine herpes virus Type 1 (BHV-1) and incubated for several time periods (24, 48, 72, 96 h). BAM were then exposed to E. coli lipopolysaccharide (LPS), or LPS with bovine serum. The amount of PCA expressed was quantified using a chromogenic assay. Viral inoculation increased bAM expression of PCA ( P < 0.01). The increase in PCA expression was larger at higher rates of viral inoculation ( P < 0.01). LPS enhanced PCA expression by bAM at low rates of viral inoculation ( P < 0.01). The effect of LPS-serum treatment was greater than the LPS alone ( P < 0.01). At high rates of viral inoculation, LPS had no enhancing effect on PCA expression. The effect of LPS on virus inoculated bAM varied with virus type, rate of inoculation, and duration of virus exposure ( P < 0.01). The results suggest that these four viruses initiate the production of PCA by bAM independently of LPS. In the field situation, an initial viral infection may induce fibrin deposition in the pulmonary alveoli prior to the establishment of a secondary gram negatmtive bacterial infection.

Resistance of naive mice to murine hepatitis virus strain 3 requires development of a Th1, but not a Th2, response, whereas pre-existing antibody partially protects against primary infection.

Abstract Murine hepatitis virus strain 3 (MHV-3) produces a strain-dependent spectrum of disease. The development of liver necrosis has been shown to be related to production of a unique macrophage procoagulant activity (PCA), encoded by the gene fgl-2, in susceptible mice. These studies were designed to examine the influence of Th1/Th2 cells on resistance/susceptibility and production of macrophage PCA in resistant (A/J) and susceptible (BALB/cJ) strains of mice following infection with MHV-3. Immunization of A/J mice with MHV-3 induced a Th1 cellular immune response, and one Th1 cell line (3E9.1) protected susceptible mice and inhibited PCA production by macrophages both in vitro and in vivo. In contrast, immunization of BALB/cJ mice with an attenuated variant of MHV-3 derived from passaging MHV-3 in YAC-1 cells resulted in a Th2 response. Transfer of spleen cells and T cell lines from immunized BALB/cJ mice failed to protect naive susceptible syngeneic mice from infection with MHV-3 and augmented macrophage PCA production to MHV-3 in vitro. However, serum from immunized BALB/cJ mice contained high titrated neutralizing Ab that protected naive BALB/cJ animals from lethal primary MHV-3 infection. These results demonstrate that susceptible BALB/cJ mice generate a Th2 response following MHV-3 infection and that these Th2 cells neither inhibit MHV-3-induced macrophage PCA production nor protect naive mice from MHV-3 infection. The results suggest that Ab protects against primary infection but cannot eradicate ongoing infection. Thus, these data define the differential role of Th1/Th2 lymphocytes in primary and secondary MHV-3 infection and emphasize the importance of PCA in the pathogenesis of MHV-3 infection.

Inhibition of Mononuclear Cell Procoagulant Activity by Lipophosphoglycan of Leishmania donovani

BACKGROUND: Since fibrin formation is an expression of the response of the host to parasite spread, the lipophosphoglycan (LPG) of Leishmania donovani and its carbohydrate fragment (PG) were examined for their capacity to inhibit procoagulant activity (PCA) production by human mononuclear cells stimulated with Escherichia coli endotoxin in vitro. METHODS: the putative inhibitory effect of LPG and its PG fragment was evaluated on the basis of their in vitro capacity to prolong significantly the time required for coagulation induced by endotoxin-stimulated mononuclear cells. RESULTS: LPG exhibited the most inhibitory activity, whereas the carbohydrate domain was not effective. These results are in agreement with the notion that LPG (but not PG) has an inhibitory effect on protein kinase C activity which plays a key role in the production of PCA by human monocytes. CONCLUSIONS: From a pathophysiological point of view, these data suggest the possibility that Leishmania avoids fibrin entrapment in the host through this inhibitory mechanism.

Procoagulant activity of bronchoalveolar lavage fluids taken from the site of tuberculous lesions.

We wanted to determine the procoagulant activity (PCA) of bronchoalveolar lavage fluids, in order to understand the macrophage-mediated lung injury at the site of tuberculous lesion. Alveolar lavage fluids taken from the site of a lesion (radiologically abnormal site (RAS)) and an unaffected site (radiologically normal site (RNS)) of active pulmonary tuberculosis (TB) patients (n = 7) and inactive (cured) patients (n = 9) were studied for their PCA producing potential. The observed results were not significant using Mann-Whitney test, and thus all increases/decreases reported below are trends/tendencies only. An increased PCA was seen in 4 out of 7 cell-free lavage supernatants of active-TB taken from the site of lesion (RAS), compared to only 1 out of 9 in inactive-TB. The PCA producing potential of the alveolar macrophages of RAS and RNS of active-TB patients was enhanced when the alveolar macrophages were co-cultured with autologous peripheral blood lymphocytes under in vitro condition. Stimulation with purified protein derivative (PPD) of M. tuberculosis showed a variable (increased or decreased) PCA production. Peripheral blood monocytes and total mononuclear cells (monocytes+lymphocytes) of active-TB patients stimulated with or without PPD showed increased PCA production, compared with normal individuals and inactive-TB patients. The present study suggests that increased production of PCA by the alveolar macrophages, in collaboration with lymphocytes and other cells at the site of tuberculous lesions will result in fibrin formation. The deposition of fibrin in the alveoli may lead to further lung injury.

Change in sensitivity to lipopolysaccharide during the differentiation of human monocytes to macrophages in vitro.

Mononuclear phagocytes in distinct differentiation stages and cultured under different conditions were tested for their sensitivity towards lipopolysaccharide (LPS), using procoagulant activity (PCA) expression and tumor necrosis factor (TNF) production as indices. The response of mature monocyte-derived macrophages differed from that of freshly isolated monocytes 1) by higher levels of constitutive PCA, 2) by responding to approximately 1,000-fold lower concentrations of LPS with PCA and TNF production, and 3) by a faster rise in PCA and TNF production. Due to the high constitutive level of PCA expression, the PCA stimulation index for LPS was low in macrophages when compared with that in monocytes. Thus, during differentiation to macrophages, human monocytes acquire increased sensitivity to LPS (2 orders of magnitude more sensitive than a sensitive turbidimetric Limulus amoebocyte lysate assay). This exquisite sensitivity to LPS is expressed regardless of whether LPS is offered in the presence or absence of lipopolysaccharide binding protein-containing serum. This points to as yet uncharacterized pathways of high affinity interaction between LPS and macrophages.

Urinary Tissue Factor Levels in Transitional Cell Carcinoma of the Bladder

Production of procoagulant activity by host and tumour cells may be increased in patients with cancer. Using a simple chromogenic assay, we have determined urinary tissue factor (TF) levels in patients presenting with transitional cell carcinoma of the bladder (TCC, n = 63), normal controls (n = 20) and patients with benign prostatic hypertrophy (BPH, n = 35). In addition, a separate cohort of patients undergoing endoscopic surveillance for superficial bladder cancer were studied to determine whether there was any difference in levels in those with recurrent disease compared to those with normal cystoscopies. Urinary TF activity was higher in TCC compared to controls (p < 0.001) and patients with BPH (p < 0.05). In patients undergoing check cystoscopy, those with recurrent disease (n = 32) had higher levels (p < 0.01) than those with normal examinations (n = 21). It is concluded that urinary TF levels are elevated in bladder cancer and that this reflects disease activity in those at risk of recurrent superficial disease. Accepted for publication February 10, 1992.

Platelet-activating factor augments tumor necrosis factor and procoagulant activity

Infusion of platelet activating factor (PAF) reproduces the host physiologic response to endotoxemia and sepsis. Tumor necrosis factor (TNF) and procoagulant activity (PCA) are two other potentially deleterious central inflammatory mediators produced in large quantities by tissue-fixed macrophages (Mφ). The relationship, if any, between PAF and TNF or PCA production is unknown. Rabbit alveolar Mφ were treated in vitro with PAF alone and prior to endotoxin (LPS). PAF alone had no effect on Mφ PCA or TNF. PAF (10 −9−10 −6 M) cotreatment enhanced Mφ PCA and TNF levels in a dose response from two- to sixfold above that of LPS treatment alone. PAF (10 −6 M) pretreatment of Mφ at T -4 to -6 hr produces an eight- to ninefold enhancement in both TNF and PCA levels. Thus, both coincubation and pretreatment or “priming” of the Mφ with PAF prior to LPS stimulation greatly increase Mφ production of PCA and TNF. The ability to augment the production of these two potent inflammatory mediators may explain in part the mechanism of action of PAF in vivo.

Platelet-activating factor primes endotoxin-stimulated macrophage procoagulant activity

Macrophage procoagulant activity (PCA) at the site of inflammation may be induced by several stimuli including bacteria and endotoxin (LPS). The local factors controlling PCA induction are poorly defined. The lipid mediator platelet-activating factor (PAF) is ubiquitous to inflammatory sites. To determine the effect of PAF on LPS-induced PCA, thioglycolate-elicited murine peritoneal macrophages were exposed to PAF (10 −7 M) or control medium for 30 min and then stimulated with LPS (10 μg/ml) for 2, 4, or 6 hr. The ability of macrophages to shorten the clotting time of plasma (ie., PCA) was then measured and clotting times were converted to PCA units using a thromboplastin standard. Cytosolic calcium ([Ca 2+] i) measurements were made using the calcium-sensitive fluorescent dye indo-1. PAF alone did not induce a rise in PCA expression (medium alone, 47 ± 11 mU/10 6 cells; PAF alone, 49 ± 12 mU/10 6 cells at t = 4 hr), but PAF treatment prior to LPS exposure resulted in a significant increase in the LPS-stimulated expression of PCA (LPS alone, 190 ± 29 mU/10 6 cells; PAF/LPS, 329 ± 57 mU/10 6 cells at t = 4 hr, P < 0.05). This priming effect was reversed by the PAF antagonist WEB 2086 (WEB/PAF/LPS, 196 ± 31 mU/2 × 10 6 cells). Stimulation of cells with PAF alone resulted in a rapid rise in [Ca 2+] i (resting, 213 ± 19 nmole; peak, 577 ± 35 nmole). This effect was also inhibited by WEB 2086. These data suggest that PAF plays an important role in the modulation of PCA production by macrophages. The rise in [Ca 2+] i stimulated by PAF may represent a cellular activation mechanism for PCA expression by peritoneal macrophages and hence may be important in fibrin deposition at inflammatory sites.

Studies on the mechanism of synthesis and release of the procoagulant activity from leukaemic cells.

The synthesis and release of procoagulant activity (PCA) from leukaemic leucocytes was studied in an in vitro culture system stimulated by endotoxin. Puromycin, actinomycin-D, vinblastine, colchicine, dibutyryl cyclic AMP and ouabain were added to the culture system to study some of the metabolic processes of these cells in relation to synthesis and release of PCA. It was found that production of PCA is an active process and depends on new protein synthesis. The release of PCA from cells can be inhibited by vinblastine, an inhibitor of microfilament and microtubules in the cell. The optimal release of PCA occurs at pH 7.2-7.4 at 37 degrees C and is not inhibited by the ATPase inhibitor ouabain. Dibutyryl cyclic AMP inhibits the release/synthesis of PCA. Gram negative septicaemia and endotoxinaemia are capable of increased production and release of PCA from leukaemic cells and could contribute to the coagulation failure seen in this disease.

In vitro and in vivo activation of endothelial cells by colony-stimulating factors.

This study was designed to identify the set of functions activated in cultured endothelial cells by the hematopoietic growth factors, granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage-colony-stimulating factor (GM-CSF), and to compare them with those elicited by prototypic cytokines active on these cells. Moreover, indications as to the in vivo relevance of in vitro effects were obtained. G-CSF and GM-CSF induced endothelial cells to proliferate and migrate. In contrast, unlike appropriate reference cytokines (IL-1 and tumor necrosis factor, IFN-gamma), G-CSF and GM-CSF did not modulate endothelial cell functions related to hemostasis-thrombosis (production of procoagulant activity and of platelet activating factor), inflammation (expression of leukocyte adhesion molecule-1 and production of platelet activating factor), and accessory function (expression of class II antigens of MHC). Other colony-stimulating factors (IL-3 and macrophage-colony-stimulating factor) were inactive on all functions tested. In comparison to basic fibroblast growth factor (bFGF), G-CSF and GM-CSF induced lower maximal proliferation of endothelial cells, whereas migration was of the same order of magnitude. G-CSF and GM-CSF stimulated repair of mechanically wounded endothelial monolayers. Exposure to both cytokines induced shape changes and cytoskeletal reorganization consistent with a migratory phenotype. To explore the in vivo relevance of the in vitro effects of these cytokines on endothelium, we studied the angiogenic activity of human G-CSF in the rabbit cornea. G-CSF, but not the heat-inactivated molecule, had definite angiogenic activity, without any sign of inflammatory reactions. G-CSF was less active than bFGF. However, the combination of a nonangiogenic dose of bFGF with G-CSF resulted in an angiogenic response higher than that elicited by either individual cytokines. Thus, G-CSF and GM-CSF induce endothelial cells to express an activation/differentiation program (including proliferation and migration) related to angiogenesis.

Tissue factor expression in endothelial cell/monocyte cocultures stimulated by lipopolysaccharide and/or aggregated IgG. Mechanisms of cell:cell communication.

A human umbilical vein endothelial cell (EC)/monocyte (MC) coculture system was used to dissect cell:cell interactions associated with production of procoagulant activity (PCA) in response to two common stimuli of intravascular coagulation in vivo (LPS and immune complexes). We found that the presence of MC at a ratio of 1 MC:10 EC increased the sensitivity of EC to LPS by 4 logs and the maximal response approximately 20-fold. Aggregated IgG alone did not stimulate the system, but in the presence of small amounts of LPS (1 to 10 ng/ml) aggregated IgG was a powerful stimulus. More than 90% of the PCA was tissue factor as shown by clotting studies and mRNA analysis. PCA was not produced by either cell alone under the conditions of study, but was produced in large amounts when the EC and MC were cocultured. The supernatant from the coculture stimulated virgin EC, but not MC, to synthesize tissue factor. The major factor in the supernatant was IL-1 beta as shown by measuring IL-1 beta, IL-1 alpha, and TNF-alpha in supernatants and by blocking the production of PCA by preincubation of supernatants with anti-cytokine antibodies. Small amounts of TNF-alpha were present in the supernatant but anti-TNF-alpha did not inhibit PCA production. Studies using recombinant cytokines established that IL-1 beta was the most potent of the cytokines tested, that cytokines potentiated each other, and that the results could be explained in quantitative terms by the amounts of IL-1 beta measured. These data emphasize that cell:cell interactions are likely to modulate procoagulant events in vivo in the presence of both LPS and immune complexes, and that IL-1 beta may be an important cytokine in these events.

Inhibitory Effect of Retinoids on the Generation of Procoagulant Activity by Blood Mononuclear Phagocytes

Retinoids are known to modulate several functions of mononuclear phagocytes. We have studied the effect of retinyl acetate (RAc) and retinoic acid (RA) on the production of procoagulant activity (PCA) by human peripheral blood mononuclear cells stimulated with endotoxin (1 microgram/ml, 4 or 20 h at 37 degrees C). Both compounds caused a dose-dependent reduction in the expression of cell-associated PCA (from 86 to less than 10% of control in the range of concentration comprised between 0.1 and 100 microM). This effect was also observed when the cells were exposed to retinoids for 10 min and washed before challenge with endotoxin, indicating that it is rapid and irreversible. In contrast, incubation of RAc or RA for 3 h at 37 degrees C with cells that have been already stimulated with endotoxin (20 h at 37 degrees C) remained without influence on cell PCA. The inhibitory action of retinoids was also observed when monocyte-enriched (greater than 85%) preparations or highly purified monocyte-derived macrophages (greater than 99%) were used instead of whole mononuclear cells. BW755C, an inhibitor of cyclo-oxygenase and lipoxygenase, reversed the inhibitory effect of retinoids, whereas acetylsalycilic acid, an inhibitor of cyclo-oxygenase, was inactive, suggesting the involvement of a lipoxygenase product. The inhibition of monocyte/macrophage PCA production and the subsequent reduction of cell potential for fibrin deposition might represent one of the mechanisms whereby retinoids exert their antiinflammatory and immunomodulatory activities.

Disparate mechanisms of induction of procoagulant activity by live and inactivated bacteria and viruses

This study describes the dose response, time course, and lymphocyte requirements of procoagulant activity (PCA) induction following stimulation of thioglycolate-elicited BALB/c peritoneal macrophages with live and inactivated bacteria (Bacteroides fragilis, Escherichia coli, and Staphylococcus aureus) and murine hepatitis virus type 3 (MHV-3). Induction of PCA by MHV-3 was significantly more rapid and the maximal PCA achieved was significantly greater than by the three bacterial species studied. In relation to induction of PCA by bacteria, the PCA response was more rapid and of greater magnitude with S. aureus and E. coli than with B. fragilis. MHV-3 induced an augmented PCA response at all concentrations of virus studied in a dose-dependent fashion, whereas higher titers of live bacteria (greater than 10(7) CFU/ml) inhibited PCA, suggesting the production of an inhibitory factor. Significant PCA induction was observed when macrophages were incubated with bacteria or virus in the absence of lymphocytes. At low titers of B. fragilis (10(3) CFU/ml), addition of lymphocytes greatly augmented PCA production, whereas at higher titers (10(7) CFU/ml), the addition of lymphocytes only slightly augmented the PCA response. In contrast, MHV-3 induction of PCA was enhanced by the addition of lymphocytes at all concentrations of virus studied, suggesting a lymphocyte-dependent process. Heat-inactivated bacteria were as effective as live bacteria in inducing PCA, suggesting that induction of PCA by bacteria requires only a bacterial surface component. In contrast, UV-inactivated MHV-3 did not induce PCA, suggesting that viral replication is a necessary step in PCA induction. These results suggest that the cellular and metabolic requirements for induction of PCA differ among viral and bacterial pathogens and may partly explain their differences in pathogenicity.

Procoagulant activity of mononuclear phagocytes from different anatomical sites in patients with gynecological malignancies

Mononuclear phagocytes exhibit complex interactions with cancer cells and might contribute to fibrin formation associated with malignancy through the production of procoagulant activity (PCA). We have studied the PCA of peritoneal macrophages in 8 patients with advanced (stages III or IV) ovarian cancer and of macrophages from regional lymph nodes in 14 patients with limited (stages I or II) uterine cancer; peritoneal and lymph-node macrophages from patients with benign gynecological tumors were used as reference cell populations. In all patients, PCA of blood monocytes was also studied. Peritoneal and lymph-node macrophages obtained from patients with ovarian and uterine cancer, respectively, expressed far higher levels of basal PCA than the corresponding cell populations from patients with benign tumors (p less than 0.001). PCA of blood mononuclear cells from patients with ovarian, but not with uterine cancer, was significantly higher (p less than 0.001) than that of control cells. High levels of D-dimer, a specific product derived from plasmin-induced degradation of stabilized fibrin, were found in all ascitic fluids and in all plasma samples but one from patients with ovarian cancer. In contrast, all controls and all uterine cancer patients but one had normal plasma D-dimer. Our findings suggest that local activation of host macrophages for PCA production might contribute to fibrin formation within the tumoral mass. In advanced cancer, blood monocytes may also be activated to produce PCA and thus contribute to activation of intravascular coagulation and, possibly, to thrombo-embolic complications frequently associated with disseminated malignancy.

Increased Macrophage Procoagulant Activity but Normal Endothelial Thrombomodulin in Rabbits Fed an Atherogenic Diet

We have studied the procoagulant activity (PCA) of blood and spleen mononuclear phagocytes and the thrombomodulin activity of aortic segments in rabbits fed an atherogenic diet for 6 weeks as compared to rabbits fed a standard diet. Blood monocytes expressed negligible basal PCA (i.e., PCA measured immediately after cell isolation) both in treated and control rabbits. PCA induced by endotoxin in vitro was not different in the two groups. In contrast, dietary treatment resulted in a significant increase in the basal PCA of spleen cells (p less than 0.01). Moreover, the latter produced significantly more PCA than control cells (p less than 0.002) in response to endotoxin in vitro. The thrombomodulin activity associated with aortic endothelium was not different in the two groups of animals despite the presence of visible fatty streaks on the aortic endothelium of treated rabbits. When rabbits were given a single injection of endotoxin, spleen mononuclear phagocytes harvested 60 min after the injection from treated animals expressed three times more PCA (p less than 0.01) than did cells from controls. In all instances PCA was identified as tissue factor. Endotoxin injection did not affect the thrombomodulin activity of thoracic aorta from both control and diet groups. It is suggested that dietary fats may cause early functional changes in mononuclear phagocytes that lead to an increased PCA production both in vivo and in vitro. These data may be relevant to an understanding of the role of monocytes-macrophages in the pathogenesis of atherosclerosis.

Production of procoagulant activity, tissue factor-like, by human mononuclear cells and its role in the pathogenesis of legionella prosthetic valve endocarditis

The production of procoagulant activity, tissue factor-like, by human mononuclear phagocytes stimulated by Legionella microorganisms is here hypothesized as a key moment in the pathogenesis of Legionella prosthetic valve endocarditis.