Effects of C5a and FMLP on Interleukin-8 Production and Proliferation of Human Umbilical Vein Endothelial Cells

Interleukin-8 (IL-8), C5a and N-formyl-methionyl-leucyl-phenylalanine (fMLP) are chemotactic peptides with predominant effects on leukocytes during inflammation. With emphasis on C5a we studied the regulation of the production of IL-8 by human umbilical vein endothelial cells (HUVEC) in vitro. Primary HUVEC cultures were incubated with IL-1, TNF, C5a and fMLP for 24 h and 48 h prior to measurement of IL-8 in supernatants of the cells by an enzyme immunoassay. Whereas IL-1 and TNF significantly increased the levels of IL-8, C5a decreased the IL-8 production after 48 h. In addition, the ability of IL-1, TNF, C5a, fMLP and IL-8 to induce cell proliferation was compared by means of a ³H-thymidine incorporation assay. In contrast with IL-1 and TNF, both C5a and fMLP increased cell proliferation of HUVEC. This increase occurred with increasing concentrations of C5a contrary to IL-8, which showed increased cell proliferation at low, but not high IL-8 concentrations.     

NADPH oxidase priming and p47phox phosphorylation in neutrophils from synovial fluid of patients with rheumatoid arthritis and spondylarthropathy

Superoxide anion (O2°-)production by neutrophil NADPH oxidase participates in arthritic joint lesion formation. Proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin 8 (IL-8) and granulocyte/macrophage-colony stimulating factor (GM-CSF) have a priming effect on neutrophil NADPH oxidase activity. NADPH oxidase activation is dependent on phosphorylation of p47phox, a cytosolic component of the enzyme. We studied O2°-production and p47phox phosphorylation in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and spondylarthropathy (SpA) according to TNF, IL-8 and GM-CSF levels. O2°-production by neutrophils isolated from SF of all the arthritis patients (RA and SpA) was higher than that of circulating resting neutrophils and when stimulated with fMLP or PMA. In addition, p47phox was partially phosphorylated in SF neutrophils compared to circulating neutrophils. High levels of TNF and IL-8 (but not GM-CSF) are detected in patient's SF (compared to circulating blood levels). TNF levels were significantly higher in RA than in SpA SF. These results suggest that increased NADPH oxidase activity could be involved in arthritic joint inflammation through increased p47phox phosphorylation. This could be the result of the presence of high levels of priming agents such as TNF and IL-8 but not GM-CSF.     

Dysregulated Cytokine Production in Human Cystic Fibrosis Bronchial Epithelial Cells

Although pulmonary inflammation is an important pathologic event in cystic fibrosis (CF), the relationship between expression of the CF gene and the inflammatory response is unclear. We studied tumor necrosis factor (TNF) and IL-1 stimulated production of IL-6 and IL-8 by CF, corrected CF, and normal human bronchial epithelial cells in culture. During the first 24 hours of TNF stimulation, CF cells produced significantly more IL-8 than normal or corrected CF cells. In the second 24 hours of TNF stimulation, IL-6 and IL-8 generation ceased in normal and corrected CF cells but accelerated in CF cells, resulting in marked IL-6 and IL-8 accumulation in CF cells. Similar results were found when cells were stimulated with IL-1. Finally, when CF cells were grown at 27°C (a culture condition which results in transport of CF transmembrane conductance regulator, CFTR, to the cell membrane and normalization of chloride conductance) TNF-stimulated production of IL-6 and IL-8 reverted to normal. We conclude that dysregulation of cytokine generation by CF bronchial epithelial cells is directly related to expression of mutant CFTR and these observations provide a potential mechanism for persistence of airway inflammation in CF.     

Monokine Production Following in Vitro Stimulation of the THP-1 Human Monocytic Cell Line with Pertussis Vaccine Components

Whole-cell pertussis found in diphtheria–tetanus–pertussis (DTP) vaccine can produce symptoms reminiscent of biological responses to circulating proinflammatory monokines such as IL-6, IL-1, and TNF. Therefore the ability of pertussis-containing vaccines and several heat-killed Bordetella pertussis preparations to stimulate cytokine production in a human monocytic cell line, THP-1, were examined. The whole-cell pertussis vaccine induced significantly more IL-6, IL-1, and TNF production than did the acellular pertussis or diphtheria–tetanus-only vaccine. Polymyxin B was able to inhibit most of the IL-6 induced by pertussis endotoxin and a heat-killed preparation of B. pertussis containing a null mutation in bvgAS, a regulatory locus required for expression of all known protein virulence factors synthesized by this organism. However, it only partially inhibited IL-6 production induced by other pertussis-containing preparations, including DTP vaccine. These results indicate that in vitro whole-cell vaccine is a potent stimulator of IL-6, IL-1, and TNF. They also suggest that although endotoxin is a major inducer of IL-6, other components of B. pertussis also contribute to IL-6 production by monocytes.     

Rho protein inhibition blocks cyclooxygenase-2 expression by proinflammatory mediators in endothelial cells

Rho proteins participate in the regulation of inflammatory gene expression in endothelial cells. We made use of Clostridium difficile toxin B-10643 (TcdB-10463) which inhibites RhoA/Rac1/Cdc42 to analyze their role in expression and regulation of cyclooxygenase-2 (COX-2) in endothelial cells (EC). Pretreatment of EC with TcdB-10643 prevented lipopolysaccharide (LPS)- or tumor necrosis factor- (TNF )-related COX-2 expression but had no effect on COX-1 protein levels. TcdB-10463 preincubation suppressed LPS-dependent nuclear factor-B activation (NF- B). Rho inhibition did not affect COX-1 activity. Inactivation of Rho proteins before LPS stimulation blocked arachidonic acid (AA)-, thrombin-, and Escherichia coli hemolysin (HlyA)-dependent release of COX-2-related 6-ketoprostaglandin F₁ (6k-PGF₁). In contrast, Rho inhibition did not affect COX-2-dependent 6k-PGF₁ liberation when TcdB-10643 was added 10 h after LPS or TNF stimulation of EC. Therefore, RhoA/Rac1/Cdc42 contribute to NF- B-dependent LPS- and TNF-induced expression of PGHS-2 in EC but had no effect on the activity of expressed COX-1 and COX-2.     

Enhancement by TNF-α of reactivation and replication of latent herpes simplex virus from trigeminal ganglia of mice

Summary The influence of tumor-necrosis-factor- (TNF-), granulocytemacrophage colony-stimulating factor (GM-CSF), interleukine-1 (IL-1) and IL-3 on the in vitro reactivation frequency and replication rate of trigeminal ganglia of mice latently infected with herpes simplex virus (HSV) strain KOS was studied. It could be demonstrated that TNF- and possibility GM-CSF, but not IL-1 and IL-3, enhanced the reactivation frequency and replication of HSV. Interferon / (IFN/) prevented reactivation and replication.     

Inhibition of PAF-, LPS-, and cytokine-induced granulocyte accumulation in guinea pig lung by dexamethasone: Evidence that inhibition of IL-5 release is responsible for the selective inhibition of eosinophilia by glucocorticoids in guinea-pigs

The potency of dexamethasone has been determined as an inhibitor of intratracheally administered platelet activating factor- (PAF) or interleukin (IL)-5-induced eosinophilia, and of lipopolysaccharide-(LPS), tumour necrosis factor -(TNF) or cytokine-induced neutrophil chemoattractant- (CINC) induced neutrophilia in guinea-pig lungs. Dexamethasone was a potent inhibitor of PAF- induced eosinophil accumulation, but higher doses of dexamethasone were required to inhibit IL-5-induced eosinophilia. LPS-induced neutrophilia was less sensitive to the inhibitory effects of dexamethasone, than PAF-induced eosinophilia. Both LPS- and TNF-induced neutrophilia were inhibited by the same doses of dexamethasone. In contrast, higher doses of dexamethasone were required to inhibit CINC-induced neutrophilia. Since data in the literature show that PAF-induced eosinophilia in guinea-pig lungs is dependent on the generation of IL-5, it is concluded that inhibition of this response, by dexamethasone, is due to inhibition of release of IL-5. Similarly, although data in the literature show that LPS-induced neutrophilia is dependent on the generation of TNF, it is concluded that inhibition of this response, by glucocorticoids, is due to an action on an event which occurs after the release of TNF, possibly through inhibition of chemokine release.accepted by G. Bowen     

Synovial activation of chondrocytes: Evidence for complex cytokine interactions

Synoviocytes secrete factors which induce the synthesis of neutral metalloproteinases (NMP) and prostaglandin E₂ (PGE₂) by chondrocytes in a response called chondrocyte activation. We analyzed synovial chondrocyte activating factors (CAF) for the presence of cytokines which modulated the NMP production by articular chondrocytes. These studies suggested the presence of several other cytokines in addition to interleukin-1 (IL-1). Both resting and activated synoviocytes contained mRNA for basic fibroblast growth factor (bFGF) which is a synergist for IL-1 induced NMP production, and secreted bFGF into their culture media. They also expressed mRNA for transforming growth factor (TGF) which inhibits IL-1 induced NMP production. These cells also produce tumor necrosis factor (TNF) and trace amounts of interleukin-6 (IL-6). In addition to these there is evidence for a synovial activator of chondrocytes which is distinct from IL-1. Since a number of recombinant cytokines including TNF, IL-6 and bFGF failed to activate chondrocytes, this could be a novel cytokine.     

Acute ethanol consumption synergizes with trauma to increase monocyte tumor necrosis factor α production late postinjury

The hypothesis that acute ethanol uptake plus trauma can synergize to increase immunosuppression was tested. We found that, unlike non-alcohol-exposed patients, patients with acute alcohol use prior to trauma have a transient decrease in monocyte tumor necrosis factor (TNF) production during the very early postinjury (0–3 days) period. However, TNF production by these alcoholexposed patients' monocytes (MØ) became hyperelevated late postinjury (>9 days). Consequently, these massively elevated MØ TNF levels can contribute to posttrauma immunosuppression after acute alcohol use. We also demonstrate that normal monocyte activation with the superantigen,Staphylococcus enterotoxin B (SEB), results in a preferential induction of cellassociated MØ TNF production, described as characteristic of immunosuppressed trauma patients. Acutein vitro ethanol treatment down-regulated the elevated TNF production by trauma patients' MØ after either SEB, muramyl-dipeptide (MDP), interferon- plus MDP, or lipopolysaccharide (LPS) stimulation. Both SEB- and LPS-induced TNF mRNA induction was inhibited by acute alcohol treatment in normal MØ, indicating that ethanol can regulate cytokine gene expression. An additional immunosuppressive effect of acute ethanol's stimulation was suggested by its induction of elevated transforming growth factor production in trauma patients' activated MØ.     

Cytokine profile in systemic lupus erythematosus,rheumatoid arthritis,and other rheumatic diseases

We investigated serum levels of interleukin-6 (IL-6), interferon-gamma (IFN-), and tumor necrosis factor alpha (TNF) from patients with systemic lupus erythematosus (SLE) and its various clinical manifestations of disease and from patients with rheumatoid arthritis (RA) and other rheumatic diseases. The serum levels of IL-6 and IFN- were highly elevated from patients with SLE associated with lymphadenopathy (LN) or nephrotic syndrome (NS). On the contrary, the serum levels of TNF were elevated from most patients with SLE associated with thrombocytopenia (TP). However, serum levels of TNF were in the normal range from patients with SLE associated with NS, LN, or central nervous system disease. Of interest, patients with SLE associated with humoral immunodeficiency disorder, hypogammaglobulinemia, had highly elevated levels of serum IL-6. The concanavalin A-stimulated mononuclear cells (MNC) of patients with SLE associated with TP secreted highly elevated levels of TNF compared to other patient groups. We suggest that abnormal production of various cytokines in SLE is an intrinsic defect of MNC and the immune system that may be the key element for a variety of clinical manifestations of this disease.     

Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

Tumor necrosis factor alpha production in schistosomiasis with carcinoma of urinary bladder

Schistosomiasis parasitic infection (Schistosoma haematobium) is associated in some patients with bladder cancer. The production of cytokines such as tumor necrosis factor alpha (TNF) is a key event of inflammation in human infectious disease and malignancy. TNF has not been previously investigated from schistosomiasis infection and bladder malignancy. In this report we demonstrate that serum levels of TNF are highly elevated in patients with schistosomiasis of urinary bladder (SB), schistosomiasis with carcinoma of urinary bladder (SCB), and carcinoma of urinary bladder without schistosomiasis (CB). Purified monocytes from bladder malignancy (SCB and CB) cultured without exogeneous stimuli release TNF in the culture supernatants. However, lipopolysaccharides and concanavalin A stimulation of monocytes from these patients produced highly elevated levels of TNF compared with normal controls. The findings that monocytes are the potent producers of TNF in this malignancy may be a key observation implicating these cells in the pathophysiology of this disease. Furthermore, it was shown that serum TNF levels correlated with the clinical staging of disease in both SCB and CB, with higher levels in T3 and T4 advanced-stage patients and low levels in T1 and T2 early-stage patients. These results suggest that monocyte abnormality and serum TNF levels might be one of the factors contributing to the progression of disease.     

GM-CSF counteracts hemorrhage-induced suppression of cytokine-producing capacity

Objective and design: To study whether a treatment with the hematopoietic growth factor GM-CSF restores the attenuated ex-vivo cytokine-producing capacity of macrophages after sublethal hemorrhagic shock.Subjects: Male Sprague-Dawley rats.Treatment: 20 g/animal of recombinant murine GM-CSF after shock via arterial line.Methods: Hemorrhagic shock was established by pressure-controlled bleeding to a mean arterial pressure of 50 mm Hg for 35–40 min and consecutive resuscitation. 24 h after hemorrhage, lipopolysaccharide (LPS)-induced cytokine production of isolated macrophages derived from different compartments was measured.Results: A significant reduction of LPS-induced TNF production was found in whole blood cultures (1.0 ± 0.7 ng/ml after sham vs. 0.23 ± 0.08 ng/ml after shock operation), macrophages derived from spleen (0.88 ± 0.23 ng/ml after sham vs. 0.03 ± 0.1 ng/ml after shock operation), peritoneum (2.2 ± 0.7 ng/ml after sham vs. 0.29 ± 0.4 ng/ml after shock operation) and bronchoalveolar fluid (0.65 ± 0.13 ng/ml after sham vs. 0.003 ± 0.027 ng/ml after shock operation, mean ± S.D.). In cells from animals treated with GM-CSF a significantly enhanced LPS-induced TNF production in splenic, alveolar and peritoneal macrophages was found after shock compared to the cells derived from untreated animals (peritoneum: 289 ± 366 ng/ml TNF after shock vs. 2066 ± 94 ng/ml TNF after shock and GM-CSF; lung: 9 ± 12 ng/ml TNF after shock vs. 64 ± 17 ng/ml TNF after shock and GM-CSF; spleen: 58 ± 96 ng/ml TNF after shock vs. 548 ± 47 ng/ml TNF after shock and GM-CSF). Blood cultures collected from rats after hemorrhagic shock did not show a significant increase of TNF-production after GM-CSF treatment.Conclusion: Hemorrhagic shock caused a depression of the TNFa response to LPS which was partly counteracted by treatment with GM-CSF. Therefore, GM-CSF represents a promising approach to normalise trauma- and shock-induced immune dysfunction.Received 4 April 2003; returned for revision 3 July 2003; accepted by A. Falus 25 August 2003     

Combined effect of hydrogen peroxide induced oxidative stress and IL-1 alpha on IL-8 production in CaCo-2 cells (a human colon carcinoma cell line) and normal intestinal epithelial cells

Oxidative stress, IL-1, and IL-8 are known to contribute to mucosal inflammation of the gastrointestinal tract. We examined the IL-8 response after brief exposure to hydrogen peroxide induced oxidative stress in CaCo-2 cells (a human colon carcinoma cell line) and in human intestinal epithelial cells. In addition, we examined whether exposure to oxidative stress, followed by IL-1, could modulate IL-8 production. A transient up-regulation of IL-8 mRNA expression was observed after hydrogen peroxide treatment. Hydrogen peroxide induced oxidative stress was also observed to promote IL-8 secretion. Exposure to hydrogen peroxide, followed by IL-1, enhanced IL-8 production over that achieved with IL-1 alone. Thus, oxidative stress and IL-1 were observed to cooperatively enhance IL-8 production.     

Regulation of ICAM-1 Expression in Mouse Macrophages

In a mouse model of silica (SI) induced lung injury, SI exposure increases expression of intercellular adhesion molecule-1 (ICAM-1) on lung (alveolar/interstitial) macrophages and alveolar type II epithelial cells. To investigate the regulation of SI induced ICAM-1 expression on mouse macrophages, freshly isolated macrophages (alveolar, peritoneal) and macrophage cell lines (MH-S, RAW 264.7) were evaluated for ICAM-1 expression elicited by the particle silica ( quartz; 20 g/ml; 6 g/cm²) or the inflammatory cytokine, TNF (20 ng/ml). TNF significantly increased ICAM-1 expression in all cell types whereas SI elicited an increase in peritoneal macrophages (PM) and the cell line, MH-S. This pattern of increased expression was confirmed by immunocytochemistry. To investigate the regulation of ICAM-1 expression, PM were incubated with SI, TNF or media concomitantly with anti-TNF antibody, the antioxidant, NAC, or the iNOS synthase inhibitor, L-NAME. Both anti-TNF and NAC, but not L-NAME, inhibited elicited (TNF, SI) as well as constitutive (media) ICAM-1 expression. These data demonstrate that both inflammatory cytokines and inorganic particles can increase ICAM-1 expression on mouse macrophages and that this expression is mediated, in part, by TNF and reactive oxygen species.     

Human α-1-acid Glycoprotein Inhibits TNF Production in the CNS of Rabbits with Meningitis: A Report of Preliminary Observations

Bacterial meningitis is accompanied by an acute inflammatory response which may be exacerbated by antibiotic treatment and subsequent killing of bacteria. Bacterial cell products induce the release of cytokines including TNF, which contribute to the inflammatory process. Alpha-1-acid glycoprotein (AAG), an acute phase reactant, is elevated during inflammation. To test whether AAG has anti-inflammatory activity we examined its effect on lipopolysaccharide-stimulated human peripheral blood mononuclear cells. Treatment of the cells with AAG in vitro resulted in reduced TNF production. To test the effects of the molecule in vivo, AAG was administered intrathecally to rabbits with Haemophilus influenzae B lysate induced meningitis. Human AAG reduced TNF production and leukocytosis in the cerebrospinal fluid. Histopathology of the leptomeninges showed markedly attenuated inflammation. These results indicate that AAG can reduce inflammation in rabbits with experimental meningitis and that the effect may be directly on TNF production by stimulated mononuclear leukocytes.     

Defective expression of FasL and Bax in human lung cancer

Abstract. The present studies investigated whether FasL and Bax genes are expressed in pleuro-pulmonary biopsies from patients with lung cancer. FasL, Bax, and TNF mRNAs were detected in 19 biopsies of primary or metastasic lung cancer by fluorescent in situ hybridization assays. Fluorescent probes were produced by polymerase chain reaction using a human spleen lambda gt11 library and specific primers for FasL, Bax, and TNF. Proteins were detected by immunohistochemistry using monoclonal anti- FasL, anti-Bax, and anti-TNF antibodies. Chromatin fragmentation was detected by TUNEL. Seven negative samples from subjects without lung pathology were obtained during legal autopsies and 12 positive control biopsies from patients with lung infections were also included. Sixty-eight percent of lung cancer biopsies exhibited FasL; Bax was expressed in 68% and TNF in 63%. FasL protein was detected in 21%, Bax protein in 26%, and TNF was present in 31% of cancer biopsies. A low degree of apoptosis in lung cancer was demonstrated by TUNEL assays. A defect in FasL, Bax, and TNF gene expression was found in lung cancer biopsies. Some tumors normally expressed the mRNA of FasL, Bax, or TNF, but their proteins were absent, or were non-functional, since TUNEL assays were negative. Such a failure would contribute to cancer cell survival and dissemination.     

Cytokine-induced neutrophil chemoattractant production by primary rat alveolar type II cells

This study was designed to determine the production of the chemokine cytokine-induced neutrophil chemoattractant (CINC) by primary rat alveolar type II (ATII) cells upon stimulation with exogenous and endogenous proinflammatory factors. Cultures of primary rat ATII cells were exposed to lipopolysaccharide (LPS), interleukin-1 beta (IL-1) or tumor necrosis factor-alpha (TNF) over a 16 hour period and the production of CINC both apically and basolaterally was measured by ELISA. Compared to unstimulated (UNS) cultures, LPS, IL-1 and TNF were found to significantly increase the level of CINC detected in culture by two, four and sixteen hours post stimulation, respectively. ATII cells also demonstrated a polar secretion of CINC. The accumulation of CINC basolaterally was significantly more than apically; 133%, 45%, 117% and 123% for UNS, IL-1, LPS and TNF respectively. We demonstrated that primary rat ATII cells may participate in the chemokine network during inflammation by the production of CINC upon stimulation with endogenous and exogenous factors.     

In situ expression of β1, β3 and β4 integrin subunits in non-neoplastic endothelium and vascular tumours

Endothelial cells play an important role in adhesive interactions between circulating cells and extracellular matrix proteins. In vitro studies have shown that many of these processes are mediated by a superfamily of heterodimeric transmembrane glycoproteins called integrins. The distribution patterns of 1, 3 and 4 integrin subunits in endothelial cells (EC) in situ were examined immunohistochemically on serial forzen sections of a wide range of non-neoplastic tissues and of vascular tumours, both benign and malignant. Expression of the 1 subunit was a constitutive feature of EC. Among the 1-associated subunits, 5 and 6 were broadly distributed in EC, irrespective of vessel size and microenvironment. The 3 subunit displayed intermediate levels of expression with a slight preference for small vessel EC. Presence of 1 was confined to EC of capillaries and venules/small veins. Expression of 2 in EC was inconsistent. With rare exceptions, the 4 chain was absent in EC. The 3 and v subunits were expressed in most EC, though not always concomitantly. In contrast to the 1 chain, however, these integrin subunits were absent in EC of glomerular capillaries and were expressed variably in sinusoidal EC. The 4 chain was evenly present in the great majority of EC, except for those of large vessels. In vascular tumours, the patterns of 1 and 1 to 6 subunit expression generally corresponded to those found in their non-neoplastic counterparts. Expression of 3, v and 4 chains, however, decreased in neoplasia, especially in angiosarcomas. These data show that EC dispose of broad and at the same time differential repertoires of integrin subunits that presumably reflect vessel-type associated functional differences among these cells. In vascular tumours, the orthologous distribution patterns of 1 and 1 to 6 chains are conserved in most instances while the amounts of 3, v and 4 subunits expressed in EC tend to decrease in the course of malignant transformation.Dedicated to Prof. Dr. med. Dres. h.c. Wilhelm Doerr on the occasion of his 80th birthday