Heritability, genetic correlation and linkage to the 9p21.3 region of mixed platelet-leukocyte conjugates in families with and without early myocardial infarction.

BACKGROUND AND AIMS: Variations in mixed platelet-leukocyte conjugate formation in human whole blood could be genetically determined. We quantified platelet and leukocyte activation and interaction in families with or without early myocardial infarction and evaluated their heritability, genetic correlation and linkage to the 9p21.3 region. METHODS AND RESULTS: The study population included 739 subjects (≥ 15 years old) from 54 large pedigrees, 23 with and 31 without familial myocardial infarction. Mixed platelet-leukocyte conjugates and markers of platelet or leukocyte activation (P-selectin, CD11b and L-selectin surface expression) were measured both before and after in vitro blood stimulation with collagen-ADP. All traits had significant genetic components (17.5-65.3% of the phenotypic variability), while shared household effects (0-39.6%) and environmental covariates (0-10.2%) tended to be smaller. Stimulated platelet-polymorphonuclear leukocyte (PMN) and platelet-monocyte conjugates showed the highest linkage to the 9p21.3 region (LOD = 0.94 and 1.33, respectively; empirical p value = 0.017 and 0.009). PMN markers resulted strongly genetically correlated between them in bivariate analysis among pairs of quantitative traits. CONCLUSION: This study supports a genetic regulation of human mixed platelet-leukocyte conjugates.

Genetic regulation of inflammation-mediated activation of haemostasis: family-based approaches in population studies.

Blood coagulation and inflammation play a key role in atherosclerosis and thrombosis. Candidate gene and genome wide association studies have identified potential specific genes that might have a causal role in these pathogenic processes. The analysis of quantitative traits is more powerful as they are closer to direct gene action than disease phenotypes. Thus linkage-based studies on extended families might be useful both to estimate the heritability and to map the genetic loci responsible for the regulation of the trait. Family-based studies may estimate high heritability for thrombosis and quantitative traits regarding both platelet aggregation and blood coagulation. Some specific loci relevant to thrombosis have been identified, with some of them showing a direct pleiotropic effect on the risk of thrombosis. Haemostasis factors can be activated by inflammatory stimuli. Fibrinogen level is genetically correlated with C-reactive protein levels with a link for both traits on chromosomes 12 and 21. Genes related to prostanoid biosynthesis, involved both in inflammation and thrombosis, show high heritability levels in both enzyme expression and prostanoid production. Considering that few large family-based linkage studies have as yet been performed on haemostasis and inflammation-related traits, additional studies are highly needed. We are performing a family-based linkage study on large pedigrees (750 subjects from 23 families with juvenile myocardial infarction and 31 control families), to identify genes responsible for quantitative traits involved in the pathway progressively going from inflammation to haemostasis, cell activation, thrombus formation and cardiovascular events.

Enhancement of mononuclear procoagulant activity by platelet 12-hydroxyeicosatetraenoic acid.

Platelets induce generation of procoagulant tissue factor activity (TFa) by mononuclear leukocytes, and also enhance the TFa induced by endotoxin. Our present investigation demonstrated that arachidonic acid, which by itself had no effect on mononuclear TFa, greatly enhanced platelet-induced TFa. The effect was concentration dependent for both platelets and arachidonate (1-20 microM); other fatty acids tested were inactive. The enhancing effect of arachidonate was more pronounced if platelets were exposed to aspirin, suggesting lipoxygenase product involvement. Production of 12-hydroxyeicosatetraenoic acid (12-HETE) was demonstrated biochemically in aspirin-treated platelet/arachidonate/mononuclear cell preparations that generated high levels of TFa. The enhancing role of 12-HETE was verified as follows. Addition of platelet-derived or synthetic 12-HETE amplified endotoxin-induced TFa more than threefold. Other lipoxygenase products were inactive. Enhancement of mononuclear cell TFa by 12-HETE represents a newly described biological function for this eicosanoid in cell-cell interactions between platelets and mononuclear cells.

Cultured human endothelial cells generate tissue factor in response to endotoxin.

Bacterial infection is associated with disseminated intravascular coagulation and fibrin deposition in the microcirculation; the mechanism of these effects in humans is still unclear. We have studied the generation of procoagulant activity (PCA) by cultured human endothelial cells (EC) in response to endotoxin. Cells from umbilical cord veins were grown in Eagle's minimum essential medium with 20% fetal calf serum till confluence. Absence of fibroblasts and macrophages was carefully checked. Endotoxin (Salmonella enteritidis lipopolysaccharide (LPS) W or Escherichia coli 0111:B4 LPS W, 0.01-1.0 micrograms/ml) was added to culture dishes for 4-6 h. PCA of EC was measured by a one-stage clotting assay and/or a two-stage amidolytic assay with the chromogenic substrate S-2222. In the absence of endotoxin, EC generated little, if any PCA (2-5 units/10(5) cells). In contrast, the addition of endotoxin resulted in generation of strong PCA that reached a maximum within 4-6 h (185-241 units/10(5) cells) and was dose-dependent between 1 and 0.01 microgram endotoxin/ml of culture medium. The generation of PCA required RNA and protein synthesis but did not require the presence of serum. No activity was found in the culture medium. The activity was of tissue thromboplastin type, as indicated by biological and immunological criteria. These endotoxin effects were observed in the absence of endothelial damage, as shown by phase-contrast microscopy and lack of 51Cr release. These data could contribute to elucidate the pathogenesis of vascular complications associated with endotoxemia in man.

Leptin induces tissue factor expression in human peripheral blood mononuclear cells: a possible link between obesity and cardiovascular risk?

BACKGROUND: Obesity is a major modifiable risk factor for cardiovascular disease. Leptin, the hormone synthesized and released primarily by adipose tissue and found increased in obese individuals, has been implicated in the regulation of inflammation and arterial and venous thrombosis. OBJECTIVE: To investigate the role of tissue factor (TF), the pivotal agonist of the clotting cascade, as a link between obesity and cardiovascular disease. METHODS AND RESULTS: In 15 obese patients, plasma levels of leptin and TF as well as TF expression in resting and endotoxin-stimulated mononuclear leukocytes (MN) were increased when compared with healthy donors. In a selected sample of obese patients, loss of body weight led to decreased circulating leptin levels, accompanied by a reduction in plasma TF as well as in TF expression, both in resting and endotoxin-stimulated MN. In subsequent in vitro experiments, leptin was incubated with MN from healthy subjects. Leptin induced TF activity and antigen in a dose-dependent fashion, as assessed by clotting assay and ELISA, respectively. Increased migration of c-Rel/p65 into the nucleus, as determined by EMSA, and development of TF mRNA in monocytes, as assessed by RT-PCR, were observed. Experiments with mitogen-activated protein kinase (MAPK) inhibitors, indicated the involvement of p38 and ERK1/2 pathways. CONCLUSIONS: The presence of TF-expressing MN in blood from obese subjects and the in vitro induction of TF by pharmacologic concentrations of leptin in MN from healthy subjects suggest that TF expression by leptin-stimulated monocytes may contribute to the cardiovascular risk associated with obesity.

Angiotensin-converting enzyme inhibitors downregulate tissue factor synthesis in monocytes.

Angiotensin-converting enzyme (ACE) inhibitors reduce the risk of recurrent myocardial infarction in patients with left ventricular dysfunction. Tissue factor (TF), the initiator of blood coagulation, plays a pivotal role in arterial thrombosis that occurs after atherosclerotic plaque fissuring. Because monocytes synthesize TF and contain several components of the renin-angiotensin system, we investigated the possibility that ACE inhibitors could modulate monocyte TF expression. Mononuclear leukocytes from healthy volunteers were incubated with endotoxin in the presence or absence of different ACE inhibitors. Captopril reduced TF expression in endotoxin-stimulated mononuclear leukocytes, as measured by a 1-stage clotting assay and ELISA analysis, by approximately 60%. The effect was dose-dependent and was attributable to ACE inhibition, given that other ACE inhibitors, such as idrapril or fosinopril, and losartan, an antagonist of the angiotensin II AT(1) receptor, caused a comparable reduction in TF activity. Reverse transcriptase-polymerase chain reaction indicated that endotoxin-mediated increased levels of TF mRNA were inhibited by ACE inhibitors. Moreover, endotoxin-induced nuclear factor-kappaB translocation to the promoter region of the gene encoding for TF was markedly inhibited by captopril. The finding that ACE inhibitors and angiotensin II AT(1) antagonists can potentially modulate TF expression by mononuclear cells has important biological and therapeutic implications for the evolution of thrombi. Our results suggest that the anti-ischemic effect of these drugs might be explained, at least in part, by their ability to reduce TF expression in monocytes.

Polymorphisms of the interleukin-1beta gene affect the risk of myocardial infarction and ischemic stroke at young age and the response of mononuclear cells to stimulation in vitro.

OBJECTIVE: To investigate the role of interleukin-1beta (IL-1beta) gene polymorphisms as a link between inflammation, coagulation, and risk of ischemic vascular disease at young age. METHODS AND RESULTS: A total of 406 patients with myocardial infarction (MI) at young age, frequency-matched for age, sex, and recruitment center, with 419 healthy population-based controls and 134 patients with ischemic stroke at young age, matched by age and sex, with 134 healthy population-based controls, were studied. Subjects carrying the TT genotype of the -511C/T IL-1beta polymorphism showed a decreased risk of MI (odds ratio [OR], 0.36; 95% CI, 0.20 to 0.64) and stroke (OR, 0.32; 95% CI, 0.13 to 0.81) after adjustment for conventional risk factors. In both studies, the T allele showed a codominant effect (P=0.0020 in MI; P=0.021 in stroke). Mononuclear cells from volunteers carrying the T allele showed a decreased release of IL-1beta and a decreased expression of tissue factor after stimulation with lipopolysaccharide compared with CC homozygotes. The presence of a monoclonal antibody against IL-1beta during cell stimulation resulted in a marked reduction of tissue factor activity expression. CONCLUSIONS: -511C/T IL-1beta gene polymorphism affects the risk of MI and ischemic stroke at young age and the response of mononuclear cells to inflammatory stimulation.

Orange juice intake during a fatty meal consumption reduces the postprandial low-grade inflammatory response in healthy subjects.

BACKGROUND AND AIMS: Evidence associates polyphenol-rich foods to reduction of low-grade inflammation and mortality for cardiovascular disease, the mechanisms underlying such effects being still unclear. Consumption of a fatty meal by healthy volunteers induces rapid and reversible low-grade inflammation. The aim of the present study was to evaluate the effect of orange juice on cellular modifications induced by a fatty meal. METHODS AND RESULTS: 18 apparently healthy subjects consumed a fatty meal, during which they drunk orange juice, either blond or red, or water, according to a randomized cross-over design. Two hours after the end of the fatty meal, both white blood cell (WBC) and platelet counts significantly increased (12.5 and 5%, respectively), while mean platelet volume decreased and a 25% release of myeloperoxidase (MPO) from polymorphonuclear leukocyte occurred. Both juices significantly prevented WBC increase and MPO degranulation, in respect to control. Triglycerides significantly increased (42%) after the fatty meal, but at a lower extent when red orange juice was consumed with the meal (20%), in respect to blond orange juice or control. This effect was statistically significant in the subgroup of 8 subjects with hypertriglyceridemia. Vascular stiffness (augmentation index), measured by Endo-PAT2000, significantly decreased after the meal only in conjunction with red orange juice. CONCLUSION: In healthy subjects the concomitant intake of orange juice may prevent the low-grade inflammatory reaction induced by a fatty meal, at cellular and possibly at vascular function levels. The relative role of different polyphenols on the observed effects of orange juices remains to be established.

Platelet-leukocyte-endothelial cell interaction on the blood vessel wall.

Leukocytes, platelets, and endothelial cells interact at sites of vascular injury and inflammation through adhesion receptors on the cell surface. On binding of ligand to receptor, these receptors initiate intracellular signaling that leads to the modulation of several biological properties of the cells involved. These finely regulated processes involve several classes of cell adhesion molecules: integrins, immunoglobulin-like proteins, selectins, and mucin-like proteins as well as an array of soluble mediators. Interaction of these cell adhesion molecules serves to recruit circulating cells to the blood vessel endothelium or to accumulated platelets on the vessel wall and to foster cell-cell communication. The importance of these interactions to inflammation, blood coagulation, and the immune response is outlined.

12-Hydroxyeicosatetraenoic acid upregulates P-selectin-induced tissue factor activity on monocytes.

12-Hydroxyeicosatetraenoic acid (12-HETE), a product of the platelet lipoxygenase pathway, amplifies tissue factor expression by P-selectin-stimulated monocytes in a time- and dose-dependent fashion. The same effect is observed when monocytes are incubated with Chinese hamster ovary cells transfected with the P-selectin cDNA. Both 5-HETE and leukotriene C4 are inactive in this system. Furthermore, the effect is not dependent on non-specific monocyte adhesion, since monocytes incubated with CHO cells expressing E-selectin do not express tissue factor, either in the presence or in the absence of 12-HETE. These results show that 12-HETE is a cofactor for the expression of tissue factor by monocytes.

Resveratrol and quercetin down-regulate tissue factor expression by human stimulated vascular cells.

BACKGROUND: Epidemiological studies have shown that consumption of wine reduces the risk of coronary heart disease. Resveratrol and quercetin, two polyphenolic compounds found in grapes and red wine, have been shown to contribute to this protection by exerting several biological properties which could be associated with cardioprotection. Tissue factor (TF), the cellular receptor that initiates blood coagulation, plays a primary role both in hemostasis following tissue injury and in the pathogenesis of atherosclerosis which predisposes to thrombosis. OBJECTIVES: We investigated the role of resveratrol and quercetin on TF expression by endothelial and mononuclear cells (MN). METHODS: Confluent human umbilical vein endothelial cells and MN collected from healthy donors were stimulated with bacterial lipopolysaccharide, interleukin-1beta or tumor necrosis factor-alpha after incubation with increasing concentrations of resveratrol or quercetin. RESULTS: In both cell types, TF activity induced by any agonist was significantly reduced by resveratrol or quercetin in a dose-dependent fashion. Northern blot analysis indicated that resveratrol and quercetin strongly reduce TF mRNA in both cell types. The inhibition of TF mRNA originated from a reduction in nuclear binding activity of the transacting factor c-Rel/p65, which was induced by the agonists and measured by electromobility shift assay. Western blot analysis revealed that the diminished c-Rel/p65 activity was dependent upon inhibition of degradation of the c-Rel/p65 inhibitory protein IkappaBalpha. CONCLUSIONS: These results provide a molecular basis which could help explain the protective activity of red wine against cardiovascular disease.

Low molecular weight fibrinogen degradation products stimulate the release of growth factors from endothelial cells.

Cultured porcine aortic endothelial cells (PAEC) constitutively produce and secrete in their culture medium mitogens collectively called endothelial cell-derived growth factors (EDGFs). Incubation of PAEC with fibrinogen-degradation products (FDPs) obtained by plasmin digestion of highly purified fibrinogen caused an increased release of EDGFs, as assessed by [3H]-thymidine incorporation in 3T3 mouse fibroblasts. The effect was time-dependent and correlated with the degree of fibrinogenolysis. It was accompanied by elongation of the cells. Neither increase in EDGFs release nor cell damage was observed when non-degraded fibrinogen was incubated with endothelial cells. Low molecular weight fibrinogen degradation products (LMWFDPs) (M(r) less than or equal to 10,000), and the higher molecular weight fibrinogen fragments D and E were tested under the same conditions. Only the LMWFDPs caused elongation and damage to PAEC and a marked stimulation (up to 12 fold) of EDGFs release. A low density growth assay revealed that the released EDGFs were mitogenically active on the same PAEC. The activity of the released EDGFs was time and dose dependent on both 3T3 fibroblasts and PAEC, indicating that LMWFDPs caused enhanced release of EDGFs that can act in paracrine and autocrine fashion. This study suggests an additional role for fibrinogenolysis contributing to wound healing, and possibly to atherosclerosis.

Cell-cell interaction and tissue factor expression.

Following tissue injury, blood components come into contact with the subendothelial tissue, a thrombogenic surface. Tissue factor, found in the media and adventitia of the vascular wall, or available on the membrane of activated monocytes and endothelial cells, triggers blood coagulation. A complex interaction between soluble molecules and cells then takes place, a fibrin mesh is formed, and the resulting clot limits or stops the loss of blood. Platelets, monocytes, and endothelial cells co-localize and interact in the area of vascular injury. This close relationship, which is regulated by an array of cell-cell adhesion molecules, favours the modulation of the biochemical pathways of these cells. The aim of this review is to summarize the contribution of these cells and their interactions in tissue factor expression and its possible relevance in the pathogenesis of vascular diseases.

Monocytes upregulate endothelial cell expression of tissue factor: a role for cell-cell contact and cross-talk.

Monocytes and endothelial cells interact at sites of vascular injury during inflammatory response, thrombosis, and development of atherosclerotic lesions. Such interactions result in modulation of several biological functions of the two cell types. Because both cells, on appropriate stimulation, synthesize tissue factor (TF), we examined the effect of human umbilical vein endothelial cell (HUVEC)/monocyte coculture on the expression of TF. We found that the coincubation resulted in TF generation, which was maximal at 4 hours, increased with increasing numbers of monocytes, and required mRNA and protein synthesis. Supernatant from HUVEC/monocyte coculture induced TF activity in HUVECs, but not in monocytes, indicating that HUVEC were the cells responsible for the activity, and that soluble mediators were involved. Interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), well-known inducers of TF in HUVECs, were found in the supernatant from the coculture, and specific antibodies directed against either cytokine inhibited TF generation. The need of IL-1 beta and TNF-alpha synthesis in order to elicit TF expression was also suggested by the delay observed in TF mRNA formation and TF activity generation when monocytes were incubated with HUVECs. IL-1 beta and TNF-alpha antigen levels in the coculture supernatant, and, consequently, HUVEC TF expression, were inhibited in the presence of anti-CD18 monoclonal antibody. These findings emphasize the role of cell-cell contact and cross-talk in the procoagulant activity, which could be responsible for the thromboembolic complications observed in those vascular disorders in which monocyte infiltration is a common feature.

Polymorphonuclear leukocytes induce PDGF release from IL-1beta-treated endothelial cells: role of adhesion molecules and serine proteases.

Polymorphonuclear leukocytes (PMNs) and endothelial cells interact at sites of vascular injury during inflammatory response and during the development of atherosclerotic lesions. Such close proximity leads to the modulation of several of the biological functions of the 2 cell types. Because we have shown previously that PMNs enhance release of growth factors from resting endothelial cells, we decided to evaluate whether coincubation of PMNs with interleukin-1beta (IL-1beta)-stimulated human umbilical vein endothelial cells (HUVEC) could further modulate mitogen release from HUVEC. We found that PMN-HUVEC coincubation resulted in a 10-fold increase in mitogen release, compared with HUVEC alone (14+/-6 versus 1.3+/-0.1). When PMNs were incubated with IL-1beta-treated HUVEC, a further increase in mitogen release (up to 35-fold) was observed. The mitogenic activity was immunologically related to platelet-derived growth factor (PDGF) because the activity was abolished by an anti-PDGF antibody. PDGF-AB antigen, detected in low concentrations in conditioned medium from HUVEC alone, was increased 4-fold when IL-1beta or PMNs were incubated with HUVEC and dramatically upregulated (up to 40-fold) when PMNs were cocultured with IL-1beta-treated HUVEC. The presence of the protease inhibitor eglin C abolished mitogenic activity generation, suggesting a role for PMN-derived elastase and cathepsin G. Indeed, purified elastase and cathepsin G mimicked PMN-induced mitogen release from HUVEC. Because PMNs firmly adhered to IL-1beta-treated HUVEC, we investigated the role of cell-cell adhesion in mitogen release. Adhesion and PDGF release were inhibited by approximately 60% in the presence of anti-CD11a/CD18 and anti-intercellular adhesion molecule-1 monoclonal antibodies. This study suggests a new role for PMNs and their interaction with endothelium in pathological conditions in which intimal hyperplasia is a common feature.

Monocytes, but not endothelial cells, downregulate the anticoagulant activity of activated protein C.

Activated protein C (APC) is a natural anticoagulant and inhibits thrombin generation by degrading factors Va and VIIIa. We evaluated the ability of APC to inhibit blood coagulation triggered by lipopolysaccharide (LPS)-stimulated [tissue factor (TF)-expressing] human mononuclear cells (MNCs) or umbilical vein endothelial cells (HUVECs). Using a plasma recalcification assay, we found that APC (up to 53.3 nmol/l final concentration) had a poor anticoagulant effect in the presence of LPS-stimulated MNCs, whereas it caused a marked prolongation of clotting time in the presence of LPS-stimulated HUVECs. A poor response to APC was also observed when platelet-free MNCs, monocyte-enriched preparations or the monocytoid cell line U937 were tested. Using a TF-independent (FXa-induced) thrombin generation assay, we demonstrated that both LPS-stimulated and unstimulated MNCs negated the inhibitory activity of APC. Direct determination of FVa activity indicated that MNCs were less efficient than HUVECs in promoting FVa inactivation by APC. Together, our results suggest that MNCs, at variance with HUVECs, protect factor Va from inactivation by APC, probably through the expression of a membrane component not present on endothelial cells. These strengthen the importance of monocytes in fibrin deposition associated with pathological conditions characterized by monocyte recruitment and activation.

Direct induction of tissue factor synthesis by endotoxin in human macrophages from diverse anatomical sites.

On exposure to endotoxin and other stimuli, human peripheral-blood mononuclear cells generate a potent procoagulant activity (PCA), identified as tissue factor. Although it is now recognized that the monocytes are the source of PCA, the question whether these cells per se are capable of procoagulant response to endotoxin or require lymphocyte collaboration remains unsettled. We have investigated the capacity of highly purified human macrophages from diverse anatomical sites to generate PCA following endotoxin stimulation. Purified (greater than 99%) monocyte-derived macrophages were obtained by prolonged (3-10 days) in-vitro culture of adherent monocytes using medium supplemented with 50% human serum. Purified (greater than 95%) peritoneal and milk macrophages were isolated by adherence to plastic. PCA was measured before and after incubation (4 hr at 37 degrees) with endotoxin (Salmonella enteritidis LPS, W or Escherichia coli O111:B4LPS, W, 1 microgram/ml final concentration) using a one-stage clotting assay and/or a two-stage amidolytic assay. Monocyte-derived macrophages had low baseline PCA (14-19 units/10(5) cells) but, upon exposure to endotoxin, displayed an eight-fold increase in PCA over control. Peritoneal and milk macrophages expressed very low baseline activity (1-5 units/10(5) cells). The latter, however, increased 15-20 times over control following endotoxin stimulation. PCA was identified as tissue factor by biological and immunological criteria. Its generation was completely abolished by cycloheximide. It is concluded that in the human mononuclear phagocyte series the capacity to produce PCA is not restricted to circulating monocytes but is also expressed by macrophages obtained from diverse anatomical sites. These macrophages appear to be autonomous in their procoagulant response to endotoxin.

Occurrence of disseminated intravascular coagulation in rat BNML leukaemia despite lack of leucocyte procoagulant activity.

Signs of disseminated intravascular clotting were observed during the development of BNML myelomonocytic leukaemia in rats, when the peripheral leucocyte count exceeded 20,000/microliters and more than 50% blasts were present in the circulation. BNML cells, harvested from blood and tested in appropriate systems, were found devoid of any procoagulant activity (PCA) even following prolonged in vitro incubation with endotoxin. Thus, it appears that these rat leukaemic cells share the same inability to express PCA which had been previously described in peripheral blood mononuclear cells from normal rats. Conceivably, in this rat model, leucocyte PCA does not represent a major trigger of intravascular coagulation and blood clotting is initiated by other, mainly plasmatic, pathways.

Role of endotoxin in the expression of human monocyte cytotoxicity.

The role of bacterial endotoxin in the expression of human monocyte cytotoxicity was studied. Endotoxin contamination of all reagents and steps of the experimental procedure were assessed by the limulus amebocyte lysate (LAL) assay. Peripheral blood monocytes were separated by adherence, and cytotoxicity was measured as [3H]-thymidine release from prelabeled mKSA-TU5 murine target cells in a 48-hr assay. Human monocytes expressed appreciable levels of spontaneous cytotoxicity under LAL- conditions irrespective of the serum source employed (LAL- or LAL+ fetal bovine serum, FBS, or LAL- human cord serum, HCS). HCS gave the highest cytotoxicity levels and LAL- FBS the lowest. Polymyxin B (10 micrograms/ml), which inhibited endotoxin-induced gelation of LAL and activation of mononuclear cells for procoagulant activity, did not affect the expression of spontaneous monocyte cytotoxicity with all three sera used. Three preparations of endotoxin used in the present study (Escherichia coli, S. typhosa, S. minnesota) caused small increases in monocyte killing in micrograms per milliliter concentrations only in 5 of 19 experiments performed. Human lymphoblastoid interferon (LAL-) and phytohemagglutinin-elicited lymphokine supernatants (LAL-) enhanced the tumoricidal activity of human monocytes under LAL- conditions and were not affected by Polymyxin B. It is concluded that exposure to endotoxin is not a prerequisite for the expression of spontaneous cytotoxicity by human blood monocytes.