von Willebrand factor biosynthesis and partitioning between constitutive and regulated pathways of secretion after thrombin stimulation

The major part of von Willebrand factor (vWf) synthesized in cultured endothelial cells is secreted constitutively without stimulation and consists of all multimeric forms of vWf. In contrast, stimulation with secretagogues such as thrombin results in the release of vWf from the storage pool, the Weibel-Palade bodies which contain only the largest, most biologically potent multimeric forms of vWf. We wished to determine whether the signal for release of vWf might also function as a signal for replenishment of the vWf by enhancing de novo biosynthesis and if replenishment of the vWf storage pool involved a diversion of newly synthesized vWf from the constitutive pathway to the regulated pathway. vWf mRNA and protein levels in unstimulated human umbilical vein endothelial cells were compared with cells that were briefly stimulated with 1 U/mL thrombin for 15 minutes and then incubated without thrombin for periods up to 72 hours. A comparison was also made between unstimulated cells and cells continuously exposed to thrombin for up to 48 hours. Thrombin stimulation, brief or continuous, had no significant effect on subsequent biosynthesis of vWf protein or vWf- specific mRNA. Since thrombin releases vWf only from the storage pool, we examined the possibility of diversion of newly synthesized vWf from the constitutive pathway to the regulated pathway. Cells were pulse- labeled, incubated for 15 minutes with and without thrombin, chased for various periods in unlabeled media, and briefly restimulated with thrombin. No significant redistribution of vWf between the two pathways was observed as a result of thrombin stimulation for the time periods tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immune activation in patients with irritable bowel syndrome

BACKGROUND AND AIMS: We set out to test the hypothesis that irritable bowel syndrome (IBS) is characterized by an augmented cellular immune response with enhanced production of proinflammatory cytokines. We further aimed to explore whether symptoms and psychiatric comorbidity in IBS are linked to the release of proinflammatory cytokines. METHODS: We characterized basal and Escherichia coli lipopolysaccharide (LPS)-induced cytokine production in peripheral blood mononuclear cells (PBMCs) from 55 IBS patients (18 mixed-, 17 constipation-, 20 diarrhea-predominant) and 36 healthy controls (HCs). PBMCs were isolated by density gradient centrifugation and cultured for 24 hours with or without (1 ng/mL) LPS. Cytokine production (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, and IL-6) was measured by enzyme-linked immunosorbent assay. Abdominal symptoms and psychiatric comorbidities were assessed by using the validated Bowel Disease Questionnaire and the Hospital Anxiety and Depression Scale. RESULTS: IBS patients showed significantly (P < .017) higher baseline TNF-alpha, IL-1beta, IL-6, and LPS-induced IL-6 levels compared with HCs. Analyzing IBS subgroups, all cytokine levels were significantly (P < .05) higher in diarrhea-predominant IBS (D-IBS) patients, whereas constipation-predominant IBS patients showed increased LPS-induced IL-1beta levels compared with HCs. Baseline TNF-alpha and LPS-induced TNF-alpha and IL-6 levels were significantly higher in patients reporting more than 3 bowel movements per day, urgency, watery stools, and pain associated with diarrhea compared with patients without these symptoms (all P < .05). LPS-induced TNF-alpha production was associated significantly (r = 0.59, P < .001) with anxiety in patients with IBS. CONCLUSIONS: Patients with D-IBS display enhanced proinflammatory cytokine release, and this may be associated with symptoms and anxiety.     

Storage of tissue-type plasminogen activator in Weibel-Palade bodies of human endothelial cells.

Tissue-type plasminogen activator (t-PA) is acutely released by endothelial cells. Although its endothelial storage compartment is still not well defined, t-PA release is often accompanied by release of von Willebrand factor (vWf), a protein stored in Weibel-Palade bodies. We investigated, therefore, whether t-PA is stored in these secretory organelles. Under basal culture conditions, a minority of human umbilical vein endothelial cells (HUVEC) exhibited immunofluorescent staining for t-PA, which was observed only in Weibel-Palade bodies. To increase t-PA expression, HUVEC were infected with a t-PA recombinant adenovirus (AdCMVt-PA). Overexpressed t-PA was detected in Weibel-Palade bodies and acutely released together with endogenous vWf by thrombin or calcium ionophore stimulation. In contrast, plasminogen activator inhibitor type 1 and urokinase were not detected in Weibel-Palade bodies after adenovirus-mediated overexpression. Infection of HUVEC with proinsulin recombinant adenovirus resulted in the storage of insulin in Weibel-Palade bodies, indicating that these organelles can also store nonendothelial proteins that show regulated secretion. Infection of AtT-20 pituitary cells, a cell type with regulated secretion, with AdCMVt-PA resulted in the localization of t-PA in adrenocorticotropic hormone-containing granules, indicating that t-PA can be diverted to secretory granules independently of vWf. Coinfection of AtT-20 cells with AdCMVt-PA and proinsulin recombinant adenovirus resulted in the colocalization of t-PA and insulin in the same granules. Taken together, these results suggest that HUVEC have protein sorting mechanisms similar to those of other regulated secretory cells. Although the results did not exclude an alternative storage site for t-PA in HUVEC, they established that t-PA can be stored in Weibel-Palade bodies. This finding may explain the acute coordinate secretion of t-PA and vWf.     

Regulation of eNOS expression in HCAEC cell line treated with opioids and proinflammatory cytokines

BACKGROUND: Nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) plays a crucial role in vascular function and homeostasis. eNOS activity maintains normal vascular tone, regulates leukocyte-endothelial cell interactions, inhibits platelet aggregation, limits smooth muscle cell proliferation and influences cardiac myocyte contractility. The loss of endothelium-derived NO has been shown to result in serious cardiovascular abnormalities, which may indicate eNOS involvement in the origin/development of cardiovascular disorders. AIM: Evaluation of eNOS mRNA level in the endothelium of human coronary arteries upon opioids treatment (mediators of ischaemic preconditioning) and after incubation with proinflammatory cytokines (stress stimuli). METHODS: Different concentrations of beta-endorphin, endomorphin-1 and endomorphin-2 (alone or in combination with the opioid receptor blocker naloxone) as well as different concentrations of cytokines alone (IL-1beta, TNF-alpha) or in combination were applied to in vitro cultured human coronary artery endothelial cells (HCAEC). After 24 hrs incubation, the cells were harvested, mRNA extracted and relative quantification of eNOS mRNA was conducted using real-time PCR. RESULTS: Opioid treatment altered eNOS expression; however, none of the changes reached a statistically significant level. As for proinflammatory cytokines, both TNF-alpha and IL-1beta substantially down-regulated the eNOS mRNA level (p <0.05). When applied in combination, these cytokines lowered eNOS mRNA even further (p <0.05). The effect was independent of the cytokine combination used. CONCLUSIONS: It was demonstrated that proinflammatory cytokines exert a substantial and statistically significant negative effect on eNOS mRNA level in human coronary artery endothelial cells in in vitro culture. Unfortunately, we were unable to demonstrate significant changes within the eNOS mRNA pool upon opioid treatment. These results indicate that opioids (basal release) do not affect eNOS expression in normal in vitro culture conditions but might do so upon stress stimuli.     

Postanoxic T lymphocyte-endothelial cell interactions induce tumor necrosis factor-alpha production and neutrophil adhesion: role of very late antigen-4/vascular cell adhesion molecule-1

The objective of this study was to define the influence of postanoxic T-lymphocyte-endothelial cell interactions on anoxia-reoxygenation (A/R)-induced neutrophil-endothelial cell adhesion and cell adhesion molecule (CAM) expression on human umbilical vein endothelial cells (HUVECs). HUVEC monolayers were exposed to 60 minutes of anoxia, followed by 24 hours of reoxygenation, wherein freshly isolated human T lymphocytes were added at 6 hours during reoxygenation. After an additional 18 hours of incubation (ie, total of 24 hours of reoxygenation), the T-cell/endothelial cell (TC/EC) coculture media were collected and added to naive HUVEC monolayers incubated with neutrophils. Although the A/R-conditioned media per se had no effect on neutrophil adhesion, the media from TC/EC cocultures significantly increased the adhesion response. This enhanced adhesive interaction was associated with significant increases in tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) levels in the TC/EC coculture media and was accompanied by a pronounced increase in endothelial E-selectin expression. Treatment of the TC/EC coculture media with anti-TNF-alpha or anti-IL-8 antibodies reduced the media-induced neutrophil adhesion response. The enhanced neutrophil adhesion and the elevated medium levels of TNF-alpha, but not IL-8, were markedly reduced by inserts that prevented direct TC/EC contact and by monoclonal antibodies directed against vascular cell adhesion molecule-1 (VCAM-1) or very late antigen-4 (VLA-4). Collectively, these findings show that VLA-4-/VCAM-1-mediated interactions between T lymphocytes and postanoxic endothelial cells stimulates TNF-alpha production, which in turn elicits endothelial cell adhesion molecule expression and a corresponding increase in neutrophil adhesion.     

TLR4-mediated inflammatory activation of human coronary artery endothelial cells by LPS

OBJECTIVE: Blood levels of cytokines are commonly elevated in severe congestive heart failure (CHF) and in coronary artery disease (CAD). While the adverse effects of cytokines on contractile function and myocardial cell integrity are well studied, little is known on whether cardiac cells are only targets or active players in these inflammatory reactions. METHODS AND RESULTS: We tested if human coronary artery endothelial cells (HCAEC) may become a source of cytokine and adhesion molecule expression when stimulated with bacterial lipopolysaccharide (LPS). Analysis of HCAEC supernatants by ELISA identified enhanced secretion of IL-6, IL-8, and MCP-1 while endothelin-1 was not increased. IL-1beta, IL-10, or TNF-alpha were not detectable by ELISA while RT-PCR revealed enhanced mRNA expression of IL-1beta and TNF-alpha but not IL-10. FACS analysis showed an LPS-induced upregulation of ICAM-1, VCAM, and ELAM-1. LFA-1 could not be detected. We further characterized receptors involved in LPS-induced signaling. Our results indicate that activation of HCAEC by LPS requires Toll-like receptor (TLR) 4. Pretreating the cells with the 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase inhibitor Cerivastatin reduced IL-6 release. CONCLUSIONS: Taken together, our results indicate that activated HCAEC may act as inflammatory cells and thus directly contribute to the progression of CHF and CAD.     

Modulation of cytokine release by purine receptors in patients with rheumatoid arthritis

OBJECTIVE: Since adenosine receptors are known to modulate the release of some inflammatory mediators in control subjects, we have examined the effects of the mixed A1 and A2 adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) on basal and lipopolysaccharide (LPS)-induced cytokine release in diluted whole blood cultures from rheumatoid arthritis (RA) patients and healthy volunteers. METHODS: Twenty-eight patients with rheumatoid arthritis aged 18-75 years gave their voluntary consent to participate and give a blood sample. Basal levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) were measured by ELISA, and whole blood cultures were prepared to assess the effects of LPS activation. RESULTS: Following a 40-hour incubation, activation of adenosine receptors by NECA, added to the cell cultures from rheumatoid arthritis patients, was found to suppress both the basal and LPS-induced release of TNF-alpha and IL-1beta, while causing an increase in the release of both basal and LPS-induced IL-6. In healthy volunteers basal cytokines were undetectable, but NECA alone induced the release of all three cytokines. Stimulated levels of TNF-alpha were more than double those in patients. In the control blood cultures, NECA suppressed LPS-induced release of TNF-alpha and IL-1beta, but increased IL-6 release. CONCLUSIONS: Adenosine receptor stimulation has a differential effect on the release of pro-inflammatory cytokines, and may induce cytokine release in normal subjects. Stimulated release of TNF-alpha is substantially lower in patients with rheumatoid arthritis than in control subjects, possibly indicating saturation, exhaustion or down-regulation of the release process.     

Tumor necrosis factor-alpha downregulates protein S secretion in human microvascular and umbilical vein endothelial cells but not in the HepG- 2 hepatoma cell line

Protein S deficiency, which is associated with thrombosis, can either be inherited or acquired. Recently, we reported that a decrease in free protein S was observed in 19 of 25 persons with HIV/AIDS. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), has been reported to be elevated in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients and has been shown to induce a procoagulant state on the surface of endothelial cells. We report here that recombinant TNF-alpha (rTNF-alpha) downregulated protein S synthesis in the SV-40T transfected human microvascular endothelial cell line (HMEC-1) model system by approximately 70% and in primary human umbilical vein and dermal microvascular endothelial cell cultures by approximately 50%. Using the HMEC-1 model, Northern blot analysis showed a decrease in protein S RNA at 24 hours that was corroborated by Western blot analysis and enzyme- linked immunosorbent assay (ELISA) quantification. Evidence supporting the specificity of the TNF-alpha effect included the following: (1) TNF- alpha down-regulation of protein S was completely blocked by TNF neutralizing antibody; (2) the effect was transient, and protein S was restored to near normal levels after TNF was removed from cell cultures; (3) an antibody directed to the TNF RI (55-kD receptor) was shown to mimic the action of TNF-alpha on HMEC-1 cells; and (4) other proinflammatory cytokines, interleukin (IL)-1, IL-6, and TGF-beta, had no effect on protein S secretion. However, TNF-alpha showed no regulatory control over protein S synthesis in the human hepatocellular carcinoma cell line HepG-2. We suggest that TNF-alpha downregulation of protein S may be a mechanism for localized procoagulant activity and thrombosis recently reported in some AIDS patients with associated protein S deficiency.     

Effect of inflammatory cytokines on the metabolism of low-density lipoproteins by human vascular endothelial cells

Cytokines have been shown to activate multiple, varied metabolic pathways in endothelial cells. Little information is available concerning the effects of inflammatory cytokines on lipoprotein metabolism by vascular endothelial cells. Human umbilical vein endothelial cells (HuECs) and bovine aortic endothelial cells (BAECs) were incubated with the inflammatory cytokines recombinant human interleukin-1beta (IL-1), tumor necrosis factor alpha (TNF), interferon gamma (gamma-IF), and interferon beta (beta-IF) at increasing concentrations (0.1 to 1,000 U/mL), for increasing periods (6 to 72 hours). After the incubation period, the media were removed and replaced with serum-free media containing radiolabeled native or acetylated low-density lipoprotein (Ac-LDL) and the rates of degradation and accumulation of radiolabeled LDL were determined. The degradation and accumulation of 125I-LDL were significantly increased (P < .02) in HuECs preincubated with IL-1 (100 U/mL) compared with control incubations without the cytokine or incubations containing gamma-IF, beta-IF, or TNF. This resulted from a 38% increase in LDL receptor protein in cells incubated with IL-1. The increased rate of LDL catabolism by HuECs incubated with IL-1 was accompanied by a significant increase (P < .05) in the rate of cholesteryl ester synthesis in the cells. Cholesteryl ester synthesis rates in HuECs preincubated with gamma-IF, beta-IF, or TNF did not differ significantly from the rates in control incubations. The effect of preincubation with cytokine on the activity of the scavenger receptor was also determined. There were no significant differences in the rate of degradation or accumulation of radiolabeled Ac-LDL in control incubations compared with cultures preincubated with IL-1, gamma-IF, beta-IF, or TNF. There also were no significant differences in the rate of catabolism of native LDL or Ac-LDL in BAECs preincubated with cytokines. Although cytokines have been shown previously to alter the binding of monocytes to endothelial cells, there was no significant increase in the binding of monocytes to cultures incubated with IL-1 plus LDL compared with IL-1 alone. In summary, we now demonstrate that cytokines, specifically IL-1, may alter LDL metabolism by human vascular endothelial cells and alter endothelial cell cholesterol metabolism. These changes in endothelial cell metabolism provide additional evidence supporting the critical role of cytokines in atherogenesis.     

Modulation of endothelial cell synthesis of von Willebrand factor by mononuclear cell products

Von Willebrand factor (vWf:Ag) is an important product of endothelial cells whose major known functions are coagulation and platelet adhesion. It appears to behave as an acute-phase reactant, levels being elevated in the plasma in a wide variety of inflammatory disorders and in the extravascular compartment in at least some of them. To determine if products of mononuclear cells, activated as part of these disease processes, may directly influence the synthesis and or release of this substance, confluent human umbilical vein endothelial cells were cultured in the presence of supernatants of mononuclear cells undergoing a mixed lymphocyte reaction, and a variety of other purified or recombinant mediators including interleukin-1 (IL-1), interleukin-2, gamma-interferon and prostaglandin E2. The endothelial cell supernatants were then assayed for vWf:Ag levels using a sensitive enzyme-linked immunoassay. The results of this study demonstrate that supernatants of a mixed lymphocyte reaction increase the synthesis and or release of vWf:Ag from endothelial cells in culture by about 100-150%. As these supernatants did not cause any increase in the release of radioisotope from 51Cr-labeled endothelial cells, it appears very unlikely that this increase was simply due to the passive release of this material from dead and dying cells. Increased vWf:Ag release was also demonstrable with purified IL-1 but not with any of the other mediators tested. These results suggest that IL-1 is able to increase the release of vWf:Ag from endothelial cells largely by an increase in synthesis and indicate at least one mechanism whereby mononuclear cells, activated during the course of an inflammatory response, may be able to influence coagulation and platelet function.     

Cytokeratin Expression and Hyaluronic Acid Production in Cultures of Human Synovial Microvascular Endothelial Cells: Influence of Cytokines and Growth Factors

Objectives: To isolate and characterize human synovial endothelial cells and to determine the effects of cytokines and fibroblast growth factor on human synovial endothelial (HSE) cell hyaluronic acid production. Methods: Endothelial cells were isolated from primary cultures of human synovial cells by fluorescent activated cell sorting based on the incorporation of a fluorescent derivative of acetylated low-density lipoprotein (DiI-Ac-LDL). Identity of endothelial cells was confirmed by positive immunostaining for von Willebrand factor (vWf), cytokeratins, endoglin, and reactivity with the lectin ulex europeaus agglutinin (UEA). Hyaluronic acid production was measured by a radioligand-binding assay. Results: HSE cells were isolated and maintained in long-term culture. The identity of the cultured cells as endothelial was based on uniform uptake of a (DiI-Ac-LDL), immunoreactivity for vWf, and endoglin and the binding of the lectin UEA. In addition, small blood vessels in the synovium were stained selectively with anticytokeratin antibodies K4.62 (cytokeratin 19 specific) and K8.13 (reactive for cytokines 1, 5, 6, 7, 8, 10, 11, and 18). Isolated HSE cells also demonstrated immunoreactivity with these cytokeratin antibodies. The cytokeratins identified by the monoclonal antibody clone K8.13 demonstrated a diffuse, fibrillar staining pattern. The cytokeratin distribution revealed with monoclonal antibody K4.62 (cytokeratin 19) was also fibrillar; however, the majority of cells also demonstrated numerous punctate cytoplasmic vesicular structures. Treatment of HSE cells with interleukin-1α (IL-1α) or acidic fibroblast growth factor (aFGF), but not tumor necrosis factor (TNFα), dramatically reduced the vesicular structures staining with the K4.62 antibody. HSE cells produced hyaluronic acid (HA) at a constitutive rate of 200–800 ng/10⁵ cells/24 h, which could be upregulated when the cells were incubated with either IL-1α or aFGF. HA production was not significantly increased when HSE cells were incubated with TNFα, IL-4 or interferon-γ. Conclusions: Synovial microvascular endothelial cells produce and secrete HA and endothelial HA secretion is upregulated by IL-1 and aFGF. IL-1 and aFGF also reduce the number of vesicular-like structures immunoreactive with a monoclonal antibody to cytokeratin 19. These studies suggest that cytokine stimulation of local endothelial secretion and/or accumulation of HA may influence leukocyte adhesion to the synovial endothelium.     

Enhanced production of tumor necrosis factor-alpha and interleukin-6 due to prolonged response to lipopolysaccharide in human macrophages infected in vitro with human immunodeficiency virus type 1

Elevated levels of circulating tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 have been detected in human immunodeficiency virus (HIV) type 1 infection. The overproduction of these cytokines could contribute to AIDS pathogenesis. Thus, the expression of TNF-alpha and IL-6 in human macrophages infected with HIV-1 was investigated. HIV-1 infection, per se, did not induce any TNF-alpha or IL-6 production or cytokine-specific mRNA expression. In contrast, HIV-1 primed macrophages to a prolonged TNF-alpha and IL-6 response to lipopolysaccharide (LPS) stimulation with respect to uninfected cells. Time-course analysis and flow cytometry demonstrated that cytokine production stopped at 6 h in uninfected macrophages but continued up to 24 h in HIV-1-infected cells. RNA studies suggested that HIV-1 interfered with late steps of cytokine synthesis. No modulation of membrane CD14 was found to account for the enhanced response to LPS. Finally, the effect of HIV-1 on cytokine response could not be abolished by the antiviral compound U75875.     

Effect of cytokines on anti-Mycobacterium avium complex (MAC) activities of human alveolar macrophages]

The macrophage is an essential component of the host defense against intracellular pathogens including MAC. Especially alveolar macrophages act as a first line defense in the lungs against MAC infection. Some cytokines were reported to activate mouse peritoneal macrophages and human monocyte-derived macrophages to inhibit growth or kill MAC. But we could find only one report describing the effect of cytokines on anti-MAC activities of human alveolar macrophages (PAM). Thus, we investigated the effect of several cytokines on anti-MAC activities of PAM. PAM were recovered from 12 healthy subjects by bronchoalveolar lavage and cells were cultured in RPMI 1640 with 10% heat-inactivated human AB serum. After 2 hours incubation nonadherent cells were discarded by vigorous washing to from monolayers of PAM (2 x 10(5) PAM in each 11-mm diameter tissue culture dish). Then we added 2 x 10(6) viable MAC bacteria (31F093T) and each cytokine to the wells simultaneously. We prepared the well without cytokines as control. After 96 hours incubation, PAM were disrupted by sonication, then all bacteria that had located inside and outside of the cells were plated onto 7H10 agar. The results are reported as mean colony forming units per each well. We had determined the optimal dose of each cytokine to prime PAM for enhanced O2- release and we used that optimal dose in this experiment. PAM with TNF-alpha pretreatment, and PAM with IFN-gamma pretreatment could release increased amount of O2- significantly, compared with control. PAM with IL-2 pretreatment and PAM with GM-CSF pretreatment also released somewhat increased amount of O2-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human serum IgA downregulates the release of inflammatory cytokines (tumor necrosis factor-alpha, interleukin-6) in human monocytes

While the protective effect of IgA antibodies against infection of the mucosal surfaces is well documented, the mechanisms involved are not entirely clear. The aim of the current study is to investigate the effect of human serum IgA on the release of inflammatory cytokines in human monocytes activated with a particulate stimulus, Haemophilus influenzae type b (Hib), or soluble lipopolysaccharide (LPS) purified from Escherichia coli. Our results show that IgA downregulates tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production, whereas IgG examined in parallel had no effect. IgA had no inhibitory effect on Hib-induced granulocyte-macrophage colony-stimulating factor release. TNF-alpha and IL-6 release were downmodulated if IgA was present during cytokine induction, and IgA was also inhibitory if added to Hib-pretreated monocytes during the phase of cytokine release. These findings indicate that there are at least two mechanisms whereby IgA antibodies can downregulate TNF-alpha and IL-6 release in human monocytes: by a mechanism acting during the time of monocyte activation, and a mechanism that downregulates the production and/or the release of these cytokines in activated monocytes. Regulation of TNF-alpha and IL-6 release by IgA may be among the antiinflammatory mechanisms preventing an uncontrolled release of potentially noxious levels of inflammatory cytokines during acute and/or chronic inflammation.     

Release of proinflammatory cytokines by mitogen-stimulated peripheral blood mononuclear cells from critically ill multiple-trauma victims

This study investigated the alterations in circulating proinflammatory cytokines and cytokine production by peripheral blood mononuclear cells (PBMCs) in response to lipopolysaccharide (LPS) or phytohemagglutinin (PHA) after severe trauma. Plasma and PBMCs were collected from 17 severely injured trauma patients and 10 healthy subjects. Plasma was stored at -80 degrees C and analyzed for cytokines. Isolated PBMCs from each subject were stimulated with LPS or PHA and incubated at 5% CO2 for 24 hours. Supernatants were collected and analyzed for cytokines. There was no significant change in the plasma concentration of free TNF-alpha and IL-1beta between healthy subjects and trauma patients. Plasma IL-6, total TNF-alpha, and total IL-1beta were significantly increased in severely traumatized patients compared with healthy control subjects. PBMCs from trauma patients produced higher levels of TNF-alpha in response to LPS but it showed no significant change in IL-1beta and IL-6 production in response to PHA or LPS in comparison to PBMCs from control subjects. We conclude that severe trauma results in a significant increase in plasma proinflammatory cytokine IL-6. Free TNF-alpha and IL-1beta in plasma remain at levels comparable to those in uninjured controls, while plasma free IL-6 levels in trauma patients remain high. Serious injury is associated with an enhanced production of TNF-alpha by PBMCs stimulated with LPS.     

IL-15 and IL-15R in leucocytes from patients with systemic lupus erythematosus

OBJECTIVE: To assess the functional status of the IL-15/IL-15Ralpha cytokine system in different leucocyte subsets from patients with systemic lupus erythematosus (SLE). METHODS: Eighteen patients with SLE (10 with inactive and eight with active disease) and 14 healthy individuals were studied. Serum levels and in vitro production of IL-15 were determined. In addition, the expression of IL-15 receptor alpha (IL-15Ralpha) and membrane-bound IL-15 was assessed and the in vitro effects of IL-15 on CD69 and CD64 expression, interferon-gamma and TNF-alpha synthesis, respiratory burst induction and apoptosis were studied. RESULTS: Serum levels of IL-15 were significantly increased in inactive and active patients with SLE. Accordingly, the in vitro synthesis and release of IL-15 by monocytes in response to IFN-gamma+lipopolysaccharide was significantly enhanced in SLE patients with active disease, as was the percentage of membrane-bound IL-15+ monocytes. On the other hand, enhanced basal expression of IL-15Ralpha was detected in leucocytes from SLE patients, with defective induction upon stimulation with phytohaemagglutinin or phorbol myristate acetate/ionomycin. Furthermore, diminished induction of CD69 expression and interferon-gamma and TNF-alpha synthesis by recombinant human IL-15 was detected in peripheral blood mononuclear cells from SLE, and there was defective induction of CD64 and priming for respiratory burst in neutrophils. The anti-apoptotic effect of IL-15 was diminished in leucocytes from SLE patients. CONCLUSION: Our data indicate that there is enhanced synthesis of IL-15 by immune cells from SLE patients, with a poor response to this cytokine by different leucocyte subsets. This abnormal function of IL-15/IL-15Ralpha may contribute significantly to the pathogenesis of SLE.     

Expression of tumor necrosis factor-alpha in cultured human endothelial cells stimulated with lipopolysaccharide or interleukin-1alpha

Tumor-necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine with a wide variety of biological effects. The most important source of this cytokine is monocytes/macrophages. It is a potent agonist in the activation of endothelial cells; however, the precise role of endothelial cells as a source of TNF-alpha is not known. In the present study, we addressed the possibility that TNF-alpha is produced by cultured human umbilical vein endothelial cells (HUVEC) stimulated with factors such as lipopolysaccharide (LPS) or interleukin-1alpha (IL-1alpha). LPS and IL-1alpha induced expression of TNF-alpha mRNA in HUVEC. IL-1alpha induced expression and secretion of TNF-alpha protein, but LPS did not induce production of TNF-alpha protein. Most of the TNF-alpha protein in cell lysate was found in the membrane fraction. The mRNA for TNF-alpha-converting enzyme (TACE) was expressed in unstimulated HUVEC, and its level was not altered by treatment with LPS or IL-1alpha. Transfection of HUVEC with full-length cDNA encoding the precursor TNF-alpha enhanced secretion of TNF-alpha protein by these cells, and treatment of the cells with a TACE inhibitor reduced the secretion. These results suggest that HUVEC produce TNF-alpha and have TACE activity. Secreted TNF-alpha may be involved in autocrine activation of endothelial cells, and TNF-alpha retained in cell membrane may serve as a juxtacrine system to activate target cells on the endothelial surface.     

Hemoglobin stimulates mononuclear leukocytes to release interleukin-8 and tumor necrosis factor alpha

Incubation of human mononuclear leukocytes (MNL) with human stroma-free hemolysate (SFH), purified adult hemoglobin Ao (HbAo), and oxidized HbAo (METHb) caused MNL to release compounds into the supernate that mediated neutrophil (polymorphonuclear leukocytes, PMN) chemotaxis and PMN adherence to human umbilical vein endothelial cells (HUVEC). Chemotaxis and PMN adherence to HUVEC were reduced significantly when supernates were preincubated with neutralizing antibodies to interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha), respectively, suggesting that IL-8 and TNF-alpha played significant roles in mediating these activities. Greatest chemotactic activity was observed in supernates of MNL treated with HbAo; while greatest PMN/endothelial cell (EC) adherence activity was observed in supernates of MNL treated with METHb. Furthermore, PMN/EC adherence activity was a function of METHb content in each hemoglobin solution. PMN chemotaxis, PMN adherence to HUVEC, and cytokine release increased as a function of increasing incubation time. Chemotactic activity was detected in HbAo- treated and METHb-treated MNL supernates after incubation for 6 hours and was maximal by 10 hours. IL-8 was detected in both HbAo and METHb- MNL supernates by 4 hours. PMN/EC adherence activity was detected in HbAo-MNL supernates at 10 hours and in METHb-MNL supernates at 4 hours. TNF-alpha was detected in METHb and HbAo-MNL supernates at 4 and 12 hours, respectively. These results suggest that hemoglobin solutions stimulate MNL to release IL-8 and TNF-alpha in quantities sufficient to induce PMN chemotaxis and PMN adherence to HUVEC. This is a US government work. There are no restrictions on its use.