Donat De Groote

Cytokine serum level during severe sepsis in human IL-6 as a marker of severity

Forty critically ill surgical patients with documented infections were studied during their stay in an intensive care unit. Among these patients, 19 developed septic shock and 16 died, 9 of them from septic shock. Interleukin 1 beta (IL-1 beta), tumor necrosis factor (TNF alpha), and interleukin 6 (IL-6) were measured each day and every 1 or 2 hours when septic shock occurred. Although IL-1 beta was never found, TNF alpha was most often observed in the serum at a level under 100 pg/mL except during septic shock. During these acute episodes TNF alpha level reached several hundred pg/mL, but only for a few hours. In contrast, IL-6 was always increased in the serum of acutely ill patients (peak to 500,000 pg/mL). There was a direct correlation between IL-6 peak serum level and TNF alpha peak serum level during septic shock and between IL-6 serum level and temperature or C-reactive protein serum level. Moreover, IL-6 correlated well with APACHE II score, and the mortality rate increased significantly in the group of patients who presented with IL-6 serum level above 1000 pg/mL. Thus, IL-6 appears to be a good marker of severity during bacterial infection.

Sepsis and serum cytokine concentrations.

OBJECTIVE To look for relationships between the classification of sepsis and plasma cytokine concentrations. DESIGN Prospective, consecutive entry study of patients meeting severe sepsis criteria and having bacteriologically documented infections. SETTING University hospital, surgical intensive care unit. PATIENTS Fifty consecutive patients developing severe sepsis or septic shock between December 1991 and December 1993. MEASUREMENTS AND MAIN RESULTS Concentrations of tumor necrosis factor, interleukin (IL)-6, IL-8, and leukemia inhibitory factor were measured by immunoradiometric assay in the plasma of patients as soon as they developed severe sepsis or septic shock. Septic shock patients were divided into three groups in a blinded fashion (i.e., without knowing the results of the concentrations of cytokines), according to the presence of sustained hyperlactacidemia and to the rapidity of the onset of sepsis. Peak concentrations of all cytokines were statistically different between severe sepsis and septic shock patients. This finding was almost exclusively due to the data from patients with rapid onset of septic shock, who demonstrated very high but transient cytokine concentrations. Septic shock patients may thus have different profiles in the time course of their cytokine concentrations. The transient, high peak concentrations of cytokines were also related to transient leukopenia. Among the cytokines measured, IL-8 appeared to be the one that correlated best with lactacidemia, the presence of disseminated intravascular coagulation, severe hypoxemia, the Acute Physiology and Chronic Health Evaluation II score, and mortality rate. CONCLUSIONS According to the profiles of the cytokines, septic shock patients do not represent a homogeneous population. These profiles should be described in order to distinguish between patients, and the profiles may be useful to identify those patients susceptible to new therapies.

Soluble tumor necrosis factor receptors in chronic hepatitis C: a correlation with histological fibrosis and activity.

BACKGROUND/AIMS Tumor necrosis factor-alpha (TNF) is a mediator of inflammation and cellular immune response. Soluble TNF receptors (sTNFR) sTNF-R55 and sTNF-R75, which compete with cellular receptors for the binding of TNF, have been detected at high levels in infectious diseases including human immunodeficiency virus and HBV infection. In order to investigate the activation of the TNF system in HCV infection, we have analyzed the balance between TNF and sTNF-R in 60 HCV-infected subjects according to their clinical, biological, virological and histological characteristics. METHODS Serum TNF, sTNF-R55 and sTNF-R75 levels were determined by ELISA before any therapy and were compared to a control group of 60 healthy subjects and a group of 34 HBV-infected patients. RESULTS Mean TNF levels were 50.5+/-4.5 pg/ml in HCV patients, and undetectable (<5 pg/ml) in the control subjects. sTNF-R55 and sTNF-R75 levels were significantly higher in HCV-infected patients than in the controls: 2.88+/-0.14 ng/ml vs. 1.30+/-0.05, (p = 0.0001), and 9.54+/-0.58 ng/ml vs. 4.19+/-016, (p = 0.0001), respectively. sTNF-R55 and TNF-alpha levels in HCV patients were not significantly different from levels in HBV patients. sTNF-R75 levels were slightly lower than in HBV patients (9.54+/-0.58 vs. 11.4+/-0.79 ng/ml, p = 0.03). In contrast to other infectious diseases, there was no correlation between levels of sTNF-R and TNF. sTNF-R75 but not TNF levels were correlated with aminotransferases levels (p = 0.0001 and p = 0.0015 for aspartate and alanine aminotransferase, respectively), while sTNF-R55 levels were significantly correlated only with aspartate aminotransferase levels (p = 0.003). sTNF-R75 levels were significantly correlated with the Metavir activity index (p = 0.01), and sTNF-R55 and sTNF-R75 levels were significantly higher in patients with vs. without cirrhosis (3.22+/-0.21 vs. 2.54+/-0.17 ng/ml (p<0.02) and 11.6+/-0.86 vs. 7.5+/-0.53 ng/ml (p<0.001), respectively). sTNF-R55, sTNF-R75 and TNF levels were not correlated with viral load, genotype or response to interferon therapy. CONCLUSIONS Levels of soluble TNF receptors, and particularly sTNF-R75, are significantly correlated with the severity of the disease but not with virological parameters such as quantitative viremia and genotype. High TNF-R production could thus suggest that HCV-related liver disease involves immunological mechanisms, including activation of the TNF system.

Clinical and biological significance of interleukin-10 plasma levels in patients with septic shock

Interleukin-10 is a potent macrophage-deactivating cytokine that inhibits lipopolysaccharide-induced tumor necrosis factor production. We determined the plasma levels of immunoreactive interleukin-10 in 16 patients with septic shock and in 11 patients with circulatory shock of nonseptic origin. In septic shock, interleukin-10 levels peaked during the first 24 h (median: 48 pg/ml) and decreased progressively till Day 5. In nonseptic shock, interleukin-10 plasma levels also increased during the first 24 h but to a lesser extent (median: 17 pg/ml). In septic shock patients, interleukin-10 plasma levels were positively correlated with tumor necrosis factor (r=0.8,p=0.01) and with parameters of shock severity including lactate levels (r=0.56, p<0.05) and correlated negatively with blood platelet counts (r=−0.65,p<0.05). The decreased production of tumor necrosis factor-α and interleukin-6 afterin vitro incubation of whole blood from septic shock patients with lipopolysaccharide was not influenced byin vitro neutralization of interleukin-10. We conclude that interleukin-10 is produced in patients with circulatory shock of septic and nonseptic origin and that the production of this anti-inflammatory cytokine during septic shock correlates positively with the intensity of the inflammatory response.

Immune monitoring in whole blood using real-time PCR

There is a need for simple and sensitive assays to assess innate and adaptive immune responses to microbial agents and vaccines. Herein, we describe a whole blood method allowing to measure the induction of cytokine synthesis at the mRNA level. The originality of this method consists in the combination of PAXgene tubes containing an mRNA stabilizer for blood collection, the MagNA Pure instrument as an automated system for mRNA extraction and RT-PCR reagent mix preparation, and the real-time PCR methodology on the Lightcycler for accurate and reproducible quantification of transcript levels. We first demonstrated that this method is adequate to measure the induction of interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1 RA) mRNA upon the addition of bacterial lipopolysaccharide (LPS) to whole blood. We then showed that this approach is also suitable to detect the production of mRNA encoding T cell-derived cytokines in whole blood incubated with tetanus toxoid as a model of in vitro immune response to a recall antigen. Finally, we demonstrated that this methodology can be used successfully to assess inflammatory as well as T cell responses in vivo, as it allowed to detect the induction of IL-1beta and IL-1 RA after injection of LPS in healthy volunteers, and also the induction of IL-2 upon recall immunisation with tetanus vaccine.

High Levels of sTNFR p75 and TNFα in Dengue-Infected Patients

Soluble forms of the two molecular species of the cell surface TNF receptors (sTNFR p55 and sTNFR p75) can reduce the activity of TNFα but they may also enhance its function by stabilizing the active TNFα oligomer. Considering the pathophysiological importance of sTNFR p75 for the regulation of the bioavailability of TNFα in the body, we determined the serum levels of sTNFR p75 and TNFα in 45 children and 28 adults with laboratory‐confirmed dengue infection by using immunoassays. The serum samples were obtained from day 1 to day 15 after the onset of the disease during the 1989–1990 outbreak of dengue‐3 in Tahiti, French Polynesia. The patients were clinically classified as having dengue hemorrhagic fever (DHF) and graded according to the criteria of the World Health Organization (WHO) into four grades from less severe (grade I) to severe (grade IV). The sera of both children and adult patients of all severity grades contained higher levels of sTNFR p75 than the sera of control subjects. Although high levels of TNFα were also detected in children and adults among grade I, II, III and IV patients, we found no correlation between sTNFR p75 and TNFα. We observed in adults a moderate elevation of sTNFR p75 and TNFα in sera compared with that observed in children. The raised levels of immunoreactive sTNFR p75 and TNFα in all clinical groups of dengue‐infected patients strongly indicate activation of the TNFα system during dengue infection. The balance between sTNFR p75 and TNFα may be altered in dengue infection. Further investigations are needed to understand the role of sTNFR p75 and TNFα in the pathogenesis of DHF and to improve the management of dengue infection.

Effects of exogenous IL-1β, TNFα, IL-6, IL-8 and LIF on cytokine production by human articular chondrocytes

Summary Cytokines are potent regulators of the chondrocyte functions. Some of them are produced by chondrocytes and interact to regulate cartilage metabolism. In this study, we investigated the production of interleukin-1 β (IL-1 β ), IL-6, IL-8 and leukemia inhibitory factor (LIF) by human chondrocytes and examined the modulation of their secretion by exogenous cytokines. Human articular chondrocytes were isolated from their extracellular matrix by a triple successive enzymatic digestion of the cartilage. Subsequently, chondrocytes were stimulated by increased amounts of human recombinant cytokines [IL-1 β , tumour necrosis factor α (TNF α ), IL-8, LIF, IL-6]. IL-1 β , IL-6, IL-8 and LIF were assayed into culture media and inside cell extracts by specific enzyme amplified sensitivity immunoassays (EASIAs). Under these experimental conditions, we have identified various interactions between cytokines. IL- β and TNF α highly stimulated IL-6, LIF and IL-8 productions. IL-6 decreased IL-8 synthesis and increased LIF production. IL-8 slightly enhanced IL-6 production. Finally, LIF stimulated IL-1 β , IL-6 and IL-8 productions. Using neutralizing antibodies against IL-1, we demonstrated that the effects of LIF were secondary to the stimulation by LIF of IL-1 β production by the chondrocytes. In conclusion, chondrocytes secrete a variety of immunocompetent cytokines including IL-1 β , IL-6, IL-8 and LIF that can interact to regulate chondrocytes metabolism. These results also define new biological activities of LIF and IL-6, and raise questions concerning their role in the pathogenesis of joint diseases.

Differential induction of monocyte chemotactic protein‐3 in mononuclear leukocytes and fibroblasts by interferon‐α / β and interferon‐γ reveals MCP‐3 heterogeneity

Monocyte chemotactic protein‐3 (MCP‐3) is a pluripotent CC chemokine, attracting most leukocytic cell types. With the use of a sensitive and specific ELISA, MCP‐3 was found to be inducible in fibroblasts and peripheral blood mononuclear cells (PBMC) by cytokines and cytokine inducers. MCP‐3 production levels (1–10 ng/ml) were tenfold lower compared to those of MCP‐1. In diploid fibroblasts, synergistic induction of MCP‐3, but not of MCP‐1, mRNA and protein was observed by combined treatment with IL‐1β and IFN‐γ. In PBMC, IFN‐α and IFN‐β (but not IFN‐γ), as well as measles virus and double‐stranded RNA, were potent inducers of MCP‐3, which suggests a role for this chemokine in an early stage of viral infections. In contrast, endotoxin failed to induce MCP‐3 production in fibroblasts and PBMC. Purification of MCP‐3 from PBMC revealed biochemical heterogeneity. In monocyte chemotaxis and calcium mobilization assays, pure 11‐kDa MCP‐3 from PBMC showed similar potencies as MCP‐3 from tumor cells. It was concluded that the induction of MCP‐3 by IFN is regulated differently in fibroblasts and PBMC. In view of the multiple target cells for MCP‐3, local and strictly regulated chemokine production might be important to conduct selectively the immune response in infection or inflammation.

Tumor Necrosis Factor Alpha Levels in Plasma and Whole-Blood Culture in Dengue-Infected Patients: Relationship Between Virus Detection and Pre-existing Specific Antibodies

The pathogenesis of dengue hemorrhagic fever (DHF) is not well known, but the role of host factors has been suggested. The level of immunoreactive circulating and cell‐generated tumor necrosis factor alpha (TNFα) was studied in 35 patients with DHF; its relationship with virus isolation and/or genome detection by reverse transcription polymerase chain reaction (RT‐PCR) and specific antibodies were detected by hemagglutination inhibition (HI). Large variation of TNFα plasma levels was obtained in dengue‐infected patients at the same stage of the disease and at the same day after infection. Most of the patients (14 out of 17 patients) who displayed augmented spontaneous in vitro production of TNFα by heparinized whole‐blood culture compared with controls also had elevated levels of TNFα in the plasma. The TNFα values in lipopolysaccharide and phytohemagglutinin heparinized whole‐blood cultures were not higher in patients than in controls, but low TNFα levels were obtained in three out of 30 patients. An inverse correlation was observed between spontaneous in vitro TNFα production and viral replication, which raises the issue of the antiviral effect of TNFα in dengue infection. The results do not support the hypothesis of the role of antibody‐dependent enhancement giving rise to increased viremic titers and production of TNFα in patients. The present study demonstrates the activation of the TNFα‐producing cells in dengue‐infected patients and suggests further investigation to define the mechanism and the role of TNFα in the pathogenesis of dengue virus infection. J. Med. Virol. 54:210–218, 1998.

Human monocytoid cell lines as indicators of endotoxin: comparison with rabbit pyrogen and Limulus amoebocyte lysate assay

The aim of this study was to develop an in vitro test system for pyrogenic substances. Three clones derived from human monocytoid cell lines, which were selected by their high sensitivity to lipopolysaccharide (LPS), were assessed for tumor necrosis factor (TNF) production. Their response to pyrogen-containing samples was compared with that in a Limulus amoebocyte lysate assay and the rabbit pyrogen test. We show here that the induction of TNF in these clones is a valid in vitro alternative to determine endotoxin in commercial preparations requiring pyrogenicity testing. Cell clones derived from Mono Mac 6 (MM6 2H8 and MM6 4B5) responded to sub-ng/ml concentrations of complete rough-strain and smooth-strain LPS, to ng/ml concentrations of diphosphoryl-lipid A, and to microgram/ml concentrations of monophosphoryl-lipid A and to detoxified LPS. Cells reacted to > or = 1 microgram/ml lipoteichoic acid by TNF production, and were relatively insensitive to toxic shock syndrome toxin-1 (TSST-1) and to muramyl dipeptide adjuvant peptide. The reaction pattern of a clone derived from THP-1 (THP-1 1G3) was in general, similar to that of the MM6 clones, except that THP-1 1G3 failed to react to diphosphoryl-lipid A. When tested on commercial samples destined for parenteral use, there was a close correlation between a sensitive Limulus amoebocyte lysate (LAL) test and the cell culture test on the one hand, and between the pyrogen test and the cell culture test on the other hand. The data suggest that this cell-based test is able to recognize pyrogens derived from gram-negative organisms in test samples with appropriate sensitivity and specificity. This test appears to be able to eliminate some of the false-positive data obtained in the LAL test.