Cytokine-induced production of IFN-beta 2/IL-6 by freshly explanted human endometrial stromal cells. Modulation by estradiol-17 beta

The cytokine IFN-beta 2/IL-6 has emerged as an important means of communication between cells--both within the immune system as well as outside it. In exploring the link between the endocrine and the immune systems, we have studied the secretion of IFN-beta 2/IL-6 by freshly explanted human endometrial stromal cells and its modulation by estrogens. Endometrial stromal cells produced IFN-beta 2/IL-6 in response to other inflammation-associated cytokines such as IL-1 alpha or beta, TNF, and IFN-gamma. This secretion was strongly inhibited by estradiol-17 beta at concentrations as low as 10(-9) M. Multiple species of stromal cell IFN-beta 2/IL-6 in the size range 23 to 30 kDa were detected using immunoprecipitation or immunoblotting procedures. The endometrial stromal cell IFN-beta 2/IL-6 species were phosphorylated and differentially glycosylated in a manner comparable to IFN-beta 2/IL-6 secreted by induced human peripheral blood monocytes or foreskin fibroblasts. However, in contrast to peripheral blood monocytes and fibroblasts, bacterial LPS did not induce IFN-beta 2/IL-6 production in endometrial stromal cells. Additionally, the IFN-beta 2/IL-6 identified in medium from IL-1 alpha-induced stromal cells is biologically active on hepatocytes. These observations, taken together with the observation that IFN-beta 2/IL-6 strongly inhibits the proliferation of human epithelial cells, suggest the possibility that stromal cell secreted IFN-beta 2/IL-6 may affect the physiology of the overlying epithelium in an hormonally modulated manner. Estrogen-regulated production of endometrial IFN-beta 2/IL-6 may participate in gender-specific systemic immunomodulation.     

Endotoxemia elicits increased circulating beta 2-IFN/IL-6 in man

beta 2-IFN/hepatocyte stimulating factor/IL-6 is a cytokine secreted by monocytes, fibroblasts, and endothelial cells in cell culture that possesses diverse biologic activity including the stimulation of acute phase plasma protein synthesis and immunomodulation. The circulating levels of this cytokine in man in response to bacterial LPS (endotoxin) were studied. A single i.v. bolus of endotoxin (20 U/kg) produced a monophasic rise in circulating immunoreactive IFN-beta 2/IL-6 and IFN-beta 2/IL-6 bioactivity (hepatocyte stimulation and B cell differentiation assays) peaking 2 to 4 h after the endotoxin challenge. Peak IFN-beta 2/IL-6 levels ranged from 4.1 to 27.5 ng/ml. Associated with this was a rise in circulating C-reactive protein levels detected 20 h after the endotoxin bolus. Thus, IFN-beta 2/IL-6 is likely one of the endogenous mediators which is triggered in man during bacterial infection and likely participates in the metabolic and immune responses of the infected host.     

IL-6/IFN-beta-2 as a circulating hormone. Induction by cytokine administration in humans

IL-6/IFN-beta 2 is a family of phosphoglycoproteins ranging in size from 19 to 30 kDa which elicits a broad range of physiologic and immune responses. Several cytokines, including TNF, have been shown to stimulate IL-6 production in cell culture. In this report, we describe the rapid induction of circulating biologically active IL-6 by the systemic administration of rTNF to patients with cancer. Low levels of IL-6 activity could be detected in the sera of patients as early as 5 min after rTNF infusion. IL-6 levels peaked approximately 2 to 3 h after rTNF bolus administration and were undetectable in most cases within 8 h. IL-6 was detected in two separate bioassays--the hybridoma B9 proliferation and the hepatocyte-stimulating factor assay. Maximum detectable levels of IL-6 ranged from 160 to 310 hybridoma growth factor units and 11-82 ng/ml in the hepatocyte-stimulating factor assay. IL-6 induction decreased after serial, daily doses of rTNF. Serial serum samples of patients receiving IL-2 or IFN-alpha were also assayed for IL-6 production. IL-2-treated but not IFN-alpha-treated patients generated low levels of IL-6 (range less than 20 to 95 hybridoma growth factor units/ml). Interestingly, in patients treated with IL-2, serum levels of TNF were detectable and peak TNF activity preceded measurable IL-6 levels. Serum levels of acute phase plasma proteins and of corticosteroid rose in response to rTNF administration. C-reactive protein increased (2.5 to 4.0-fold) within 8 h of rTNF administration and cortisol levels rose (10- to 20-fold) within 4 h after rTNF injection. We conclude that rTNF administration in man leads to the induction of circulating IL-6 which, due to its broad range of activities, may be an important physiologic signal regulating the immune response.     

Interleukin-6 in neuro-Behçet’s disease: Association with disease subsets and long-term outcome

Increased cerebrospinal fluid (CSF) IL-6 has been reported in patients with Behçet’s disease (BD) and neurological involvement. To elucidate the value of IL-6 as a marker of disease activity, serum and CSF IL-6 levels of 68 BD patients with acute (26) or chronic progressive (14) parenchymal involvement (pNB), dural sinus thrombosis (10), ischemic stroke (5) or headache (13) were measured by ELISA. Samples from multiple sclerosis, subacute sclerosing panencephalitis, and noninflammatory neurological disorders were used as controls. CSF but not serum samples of neuro-BD patients with acute pNB displayed significantly increased IL-6 levels as compared to other groups. Chronic progressive pNB patients also showed increased CSF IL-6 levels, albeit less prominent. Patients with increased CSF IL-6 levels were more likely to have increased CSF cell counts and total protein levels and these three parameters were correlated with long-term (3 years) disease outcome. In four chronic progressive patients, IL-6 was elevated despite otherwise normal CSF. CSF IL-6 seems to be a marker of disease activity and long-term outcome for pNB along with CSF cell count and protein levels. CSF IL-6 could be used in chronic progressive patients who have normal CSF cell, or protein levels to detect disease activity.     

IL-6/IFN-beta 2 in synovial effusions of patients with rheumatoid arthritis and other arthritides. Identification of several isoforms and studies of cellular sources

We have looked for IL-6, a cytokine that has immunomodulating and inflammation-associated activities, in joint exudates (fluid and mononuclear cells) from patients with rheumatoid arthritis and other arthritides using both biologic and biochemical assays. IL-6 was assessed by its ability to stimulate alpha 1-antichymotrypsin secretion from the human hepatoma cell line Hep3B clone 2, an activity which is blocked by an antiserum to Escherichia coli derived IL-6, and by the growth of the IL-6-dependent murine hybridoma 7TD1 cell line. IL-6 isoforms in synovial fluid were characterized by immunoaffinity chromatography followed by Western blotting. The presence of IL-1 in synovial fluids and its production by synovial fluid mononuclear cells was monitored by Western blotting and indirect immunofluorescence with polyclonal anti-IL-1 beta antisera. In an analysis of 30 effusions from 27 rheumatoid patients with acutely inflamed joints, abundant quantities of IL-6 (greater than 2 ng/ml) were detected in 23 by the alpha 1-antichymotrypsin bioassay. Several rheumatoid synovial fluids also had elevated IL-6 levels in the 7TD1 bioassay. Seven of nine nonrheumatoid effusions also contained high levels of IL-6 (greater than 2 ng/ml). No IL-1 (less than 0.25 ng/ml) could be detected by Western blotting in 10 rheumatoid effusions even though eight of these contained high levels of IL-6. The IL-6 activity could be neutralized with a rabbit antiserum to rIL-6. Multiple IL-6 isoforms (25, 30, 45 kDa) were present in two rheumatoid and one traumatic effusion studied. Fresh mononuclear cells isolated from various synovial effusions did not appear to make IL-6 constitutively, as no IL-6 could be detected in the media of cells cultured for 12 to 18 h after isolation. Similarly, there was no constitutive production of IL-1 by these cells. However, synovial fluid mononuclear cells could be induced to secrete both IL-6 and IL-1 after stimulation with LPS. The LPS-responsive cells were monocytes and not lymphocytes or dendritic cells. These findings suggest that IL-6 is involved in inflammatory joint disease. However, the primary cells synthesizing it may be located in the synovial lining instead of the joint exudate.     

Caspase-1 levels in biological fluids from patients with multiple sclerosis and from patients with other neurological and non-neurological diseases

In multiple sclerosis (MS), pathological white matter damage in the central nervous system is sustained by immune-inflammatory response. Caspase-1 plays a pivotal role in immune-mediated inflammation, as it regulates the cellular export of IL-1beta and IL-18. We carried out a preliminary in vitro study of the kinetics of extracellular caspase-1 release. We then measured caspase-1 levels in paired serum and cerebrospinal fluid (CSF) samples of 75 MS patients, 15 healthy subjects, and patients with other neurological diseases. Paired synovial fluid and serum samples of patients with juvenile idiopathic arthritis, and paired sputum and serum samples of asthma patients were also studied. Mean serum caspase-1 concentrations did not differ between groups. Caspase-1 was detected in the CSF of patients with acute, but not stable, MS [7.5 +/- (SEM) 0.9 pg/ml; test's sensitivity, 56% and specificity, 100%]. Its levels correlated with pleocytosis. The highest mean caspase-1 levels were found in the arthritic synovial fluids (945.5 +/- 126.6 pg/ml, which correlated with erythrocyte sedimentation rate), and in the sputum samples (370.1 +/- 71.0 pg/ml, which correlated with the number of macrophages in the sputum). On condition that caspase-1 is determined in the fluids pertaining to the disease-specific inflammatory sites, its level is a reliable marker of ongoing immune-inflammatory response. The enzyme measurement in CSF can also help define state-trait in MS.     

A Folate Binding Protein in Ascitic Fluid, Serum and Ovarian Tissue of Patients with Ovarian Adenocarcinoma Immunoreacts with Antibodies Against Human Milk Folate Binding Protein

The presence of a folate binding protein which immunoreacts with antibodies against human milk folate binding protein was demonstrated in ascitic fluids from seven patients with ovarian adenocarcinoma. Ascitic fluids collected from two patients with other malignancies contained non-immunoreactive FBP. Tumor tissue specimens from five patients with ovarian carcinoma contained immunoreactive FBP. By contrast to normal ovaries ovarian carcinoma tissue showed positive immunostaining on immunohistochemistry. Ascitic fluids from two patients with ovarian carcinoma exhibited single distinct bands on SDS-PAGE immunoblotting. The gel filtration profile of ovarian carcinoma tissue homogenate from two patients contained 25 and 100 kDa peaks of radioligand-bound and immunoreactive folate binding protein, while ascitic fluid from one of the patients exhibited a large 100 kDa immunoreactive peak with no radioligand binding activity. The immunoreactive non-functional 100 kDa FBP could represent unprocessed precursor FBP. Future studies are necessary to evaluate whether determination of immunoreactive FBP in ovarian adenocarcinomatosis is of any diagnostic value.     

Suppression of IL-2-induced SAA gene expression in mice by the administration of an IL-1 receptor antagonist

The hepatic acute phase response induced by the administration of interleukin (IL)-2 is most likely mediated by secondary cytokines. In this investigation, we examined the role of endogenous IL-1 in the synthesis of the hepatic acute phase protein serum amyloid A (SAA) during IL-2 treatment. The injection of IL-2 induced SAA gene expression in the liver. The concurrent administration of an IL-1 receptor antagonist (IL-1RA) markedly reduced hepatic SAA mRNA levels and, to a lesser extent, SAA protein levels in the serum. Although IL-1 is an inducer of IL-6 production, the administration of the IL-1RA had no effect on circulating IL-6 levels in IL-2-treated mice. These findings suggest that the production of IL-1 is an important factor in the induction of SAA mRNA in mice undergoing immunotherapy with IL-2.     

A cytokine-selective defect in interleukin-1 beta-mediated acute phase gene expression in a subclone of the human hepatoma cell line (HEPG2)

Several well-differentiated human hepatoma cell lines (HepG2, Hep3B) have been used to identify factors which regulate hepatic gene expression during the host response to inflammation/tissue injury (acute phase response). Studies in these cell lines, as well as in primary cultures of rat, rabbit, and mouse hepatocytes, have demonstrated that interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF-alpha), and interferon-beta 2 (IFN-beta 2) each mediate changes in expression of several hepatic acute phase genes. In this study we identify a subclone of the HepG2 cell line in which there is a selective defect in IL-1 beta-mediated acute phase gene expression. Recombinant human IL-1 beta mediates an increase in synthesis of the positive acute phase complement protein factor B and a decrease in synthesis of negative acute phase protein albumin in the parent uncloned HepG2 cell line (HG2Y), but not in the subclone HG2N. Recombinant human IFN-beta 2 and TNF-alpha, however, regulate acute phase protein synthesis in the subclone HG2N; i.e. IFN-beta 2 and TNF-alpha increase synthesis of factor B and decrease synthesis of albumin in both HG2Y and HG2N cells. Equilibrium binding analysis with 125I-rIL-1 beta at 4 degrees C showed that both HG2N and HG2Y cells bind IL-1 beta specifically and saturably. HG2N and HG2Y possess 3.8 and 4.0 x 10(3) plasma membrane receptors/cell with affinities of 0.96 and 1.07 x 10(-9) M, respectively. Thus, the defect in this subclone of the HepG2 cell line is likely to involve the signal transduction pathway for the biological activity of IL-1 beta and will be useful in elucidation of this signal transduction pathway.     

Production of random classes of immunoglobulins in brain tissue during persistent viral infection paralleled by secretion of interleukin-6 (IL-6) but not IL-4, IL-5, and gamma interferon

The activities of cytokines were determined in cerebrospinal fluid (CSF) and serum of mice persistently or intracerebrally acutely infected with lymphocytic choriomeningitis (LCM) virus (LCMV). In contrast to CBA/J (LCMV carrier) mice that responded with low levels of LCMV-specific antibody, high-responder NMRI (carrier) mice showed antibody production by B cells outside of lymphoid organs. The B cells that had infiltrated the brains of LCMV carrier mice exhibited no preferential immunoglobulin isotype or subtype virus-specific antibody production. Phenotypic analysis of the brain infiltrates in virus carrier mice revealed dominance of CD4+ T cells in contrast to virtual absence of CD4+ and dominance of CD8+ in mice with acute LCM. In NMRI but not in CBA/J carrier mice, significant concentrations of interleukin-6 (IL-6) were detected in CSF and serum; IL-2, IL-4, IL-5, granulocyte-macrophage CSF (GM-CSF), and gamma interferon (IFN-gamma) were not elevated. In contrast, during acute, lethal LCM, IL-6 and IFN-gamma were found at high concentrations, and IL-4, IL-5, and GM-CSF were detectable in CSF and serum, but virus-specific antibody-producing cells were not (yet) detectable in the brain. Thus, distinct cytokine patterns are found in acute versus chronic LCMV infection of the brain: in LCM carrier mice, local random-class immunoglobulin production correlated with the absence of IL-2, IL-4, IL-5, and IFN-gamma but active secretion of IL-6.     

Beneficial effect of interferon-beta treatment in patients with multiple sclerosis is associated with transient increase in serum IL-6 level in response to interferon-beta injection

In order to predict the clinical benefit of interferon-beta (IFN-beta) to patients with multiple sclerosis (MS), the following markers were investigated; (1) chronological change of cytokines (IFN-gamma, TNF-alpha, IL-6, IL-10, and TGF-beta) after administration of IFN-beta, (2) untoward effects of IFN-beta such as headache and arthralgia, (3) backgrounds of the patients such as age and relapse rate, (4) efficacy of IFN-beta therapy assessed by the change of relapse rate and progression of disability. Chronological blood sampling was performed 0, 10, and 24 h after injection of IFN-beta. The increase of serum IL-6 level in response to IFN-beta administration was associated with headache, arthralgia, relapse rate before treatment, and disability score at the initiation of the therapy. Significant association of change of serum TNF-alpha with age and headache was also observed. The important finding in this study was that patients with a transient increase in IL-6 in response to IFN-beta showed a slow disease progression. This result suggests that this transient increase in the serum IL-6 predicts favorable response to IFN-beta treatment.     

Interferon-beta 2/interleukin 6 is a co-stimulant for human T lymphocytes

IFN-beta 2/IL-6 promotes the proliferation of human peripheral blood T cells in the presence of either PHA or Con A. This effect is observed with highly purified T cells and is masked by the addition of as few as 2% monocytes, suggesting that accessory cells are not required and that IFN-beta 2/IL-6 acts directly on the T cells. Both T4-positive and T8-positive cells respond to IFN-beta 2/IL-6 plus PHA. Studies of the time requirements of IFN-beta 2/IL-6 and of PHA in the response of T cells show that optimal co-stimulation occurs when both IFN-beta 2/IL-6 and PHA are added together at the outset of culture, suggesting that IFN-beta 2/IL-6 acts predominantly on resting T cells. Unlike T cell proliferation induced by IL-2, T cell proliferation induced by IFN-beta 2/IL-6 is not blocked by antibodies to the IL-2 R, suggesting that IFN-beta 2/IL-6 does not act by stimulating IL-2 production. Thus, in addition to its previously reported properties, IFN-beta 2/IL-6 stimulates T cells in the presence of mitogen, and may prove to be of considerable importance in the physiologic activation of T cells.     

IFN-beta 2, B cell differentiation factor 2, or hybridoma growth factor (IL-6) is expressed and released by human epidermal cells and epidermoid carcinoma cell lines

IL-6, which is also known as IFN-beta 2, hybridoma growth factor, hepatocyte-stimulating factor, and B cell differentiation factor, mediates acute phase responses including fever, has lymphocyte-stimulating capacities, and antiviral activity. IL-6 is produced by monocytes, fibroblasts, certain lymphocytes, and various tumor cells. The present study demonstrates that this multifunctional cytokine is released also by normal human epidermal cells (EC) and human epidermoid carcinoma cell lines (A431, KB). Accordingly, supernatants derived from freshly isolated EC, long term keratinocyte cultures, A431, or KB cells stimulated the proliferation of a hybridoma growth factor/IL-6-dependent plasmacytoma cell line (B9). IL-6 constitutively was produced in the presence of serum proteins. The addition of IL-1 alpha, IL-1 beta, or the tumor promoter PMA significantly enhanced the synthesis and release of EC-derived IL-6 (EC-IL 6). Like monocyte or fibroblast-derived IL-6, EC-IL-6 exhibited Mr microheterogeneity within 21 and 28 kDa. Similarly in Western blotting experiments an antiserum directed against human rIFN-beta 2/IL-6 detected the different Mr forms of EC-IL-6. Moreover, this antiserum was able to block the B9 cell growth-promoting capacity of EC-IL-6 strongly suggesting that this EC-derived mediator is closely related, if not identical with IL-6. This was further confirmed by Northern blot analysis detecting IL-6 specific mRNA both in long term cultured keratinocytes and A431 cells by hybridization with a cDNA fragment encoding for B cell differentiating factor 2/IL-6. Therefore, in addition to the production of other cytokines as previously reported, EC and in particular keratinocytes also synthesize and release IL-6. This further supports the important regulatory role of the epidermis during the pathogenesis of inflammatory, autoimmune, and neoplastic diseases.     

Compartmentalization of TNF and IL-6 in meningitis and septic shock

We examined the compartmentalization of bioactive tumour necrosis factor (TNF) and interleukin 6 (IL-6) to the subarachnoid space and systemic circulation in patients with meningococcal meningitis and septic shock/bacteraemia. In patients with meningitis, median levels of TNF in 31 paired samples of cerebrospinal fluid (CSF) and serum were respectively 783 pg/ml and below detection limit (p < 0.001) and median levels of IL-6 were 150 ng/ml and 0.3 ng/ml (p < 0.0001). In patients with septic shock without meningitis, median levels in paired samples of CSF and serum were respectively below detection limit and 65 pg/ml (not significant, (ns)) (TNF, eleven patients) and 1.3 ng/ml-3 ng/ml (ns) (IL-6, nine patients). The data show that TNF and IL-6 are localized to the subarachnoid space in patients with meningitis although the blood-brain barrier is penetrable to serum proteins. On the other hand, patients with septic shock tend to have cytokines in both serum and CSF.     

Analysis of serum and cerebrospinal fluid cytokine levels in subacute sclerosing panencephalitis in Papua New Guinea

BACKGROUND: Subacute sclerosing panencephalitis (SSPE) is a rare progressive inflammatory disease characterized by the persistent infection of the brain by the measles virus. However, the immunological pathophysiology of SSPE is still unclear. METHODS: We measured the concentrations of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-4, IL-6, IL-10, and soluble TNF receptor 1 (sTNFR1) in the serum and cerebrospinal fluid (CSF) of 23 patients with SSPE in Papua New Guinea (PNG), a country with a high incidence of SSPE, and Japanese controls by cytometric bead array or ELISA. RESULTS: The serum IL-6 and IL-10 levels of SSPE patients were significantly higher than those of controls (p=0.0075, and p=0.0019, respectively). The serum IL-6 and IL-10 levels of SSPE patients with fever were significantly higher than those without fever (p=0.0107, and p=0.0006, respectively). The CSF IL-6 levels of SSPE patients were significantly higher than those of controls (p=0.0218). The CSF IL-6 levels of SSPE patients with myoclonic jerks were significantly higher than those without myoclonic jerks (p=0.0189). There were no differences in serum IFN-gamma, TNF-alpha, IL-2, IL-4, and sTNFR1, or CSF IFN-gamma, TNF-alpha, IL-2, IL-4, IL-10, and sTNFR1 levels between the affected patients and controls. CONCLUSION: Our present study suggests that serum IL-6 and IL-10 levels are related to fever, and the CSF IL-6 level, myoclonic jerks, in SSPE patients in PNG.     

Microassay of inorganic sulfate in biological fluids by controlled flow anion chromatography

The application of controlled flow anion chromatography to the assay of inorganic sulfate in biological fluids is described. The sulfate anion is separated from other anions by ion-exchange chromatography and quantitated conductimetrically. Coefficient of variance is 3.4%, about half that for the barium precipitation assay. Interference from heparin in plasma samples and unknown sources in tissue extract analysis is avoided. Sulfate levels in plasma are not different from those measured in serum after protein precipitation. Normal levels for sulfate concentration in human plasma, cerebrospinal fluid and hepatic tissue extract are reported.     

Interleukin-1 receptor antagonist, soluble tumor necrosis factor-alpha receptor type I and II, and soluble E-selectin serum levels in multiple sclerosis patients receiving weekly intramuscular injections of interferon-beta1a

BACKGROUND: interferon beta (IFN-beta) reduces relapse rate and disease progression in patients with the relapsing-remitting form of multiple sclerosis (RRMS). IFN-beta may act by upregulating the expression of anti-inflammatory components of the immune system. OBJECTIVES: To determine whether weekly intramuscular (i.m.) injection of IFN-beta1a had a short- or long-term effect on the expression of naturally occurring soluble factors that play an immunosuppressive role within the cytokine network. MATERIALS AND METHODS: serum levels of interleukin-1 receptor antagonist (IL-1Ra), soluble tumor necrosis factor alpha receptor type I and type II (sTNF-alphaRI and sTNF-alphaRII), and soluble E-selectin (sE-Sel) were followed over time in ten patients with RRMS who were treated with weekly i.m. injections of 30 mg (= 6 MU) of IFN-beta1a. Patient sera were sampled before, and 24, 48, 72, 96, and 168 hours after the first IFN-beta1a injection (short-term), and then at 1, 3, 6, 9 and 12 months after therapy initiation (long-term); highly sensitive, commercially available ELISA tests were used. RESULTS: serum levels of IL-1Ra, sTNF-alphaRI and sTNF-alphaRII, but not sE-Sel were significantly increased in both short- and long-term follow-up. Interestingly, IL-1Ra, sTNF-alphaRI and sTNF-alphaRII behaviors were completely different, suggesting that these naturally occurring immunoregulatory factors were differentially affected by IFN-beta1a. CONCLUSION: our study demonstrates that weekly i.m. injection of 30 mg of IFN-beta1a induces the expression of soluble mediators that may suppress the activities of pro-inflammatory cytokines such as IL-1 and TNF-alpha.     

Cytokine production after intravenous or peritoneal gram-negative bacterial challenge in mice. Comparative protective efficacy of antibodies to tumor necrosis factor-alpha and to lipopolysaccharide.

The production of TNF-alpha, IL-1, and IL-6 was measured in mice after bolus i.v. Escherichia coli O111 LPS injections and during bacteremia induced either by bolus i.v. or by i.p. challenges of live E. coli O111. High but transient TNF-alpha peaks were observed after bolus i.v. LPS or bacterial challenges. In contrast, the levels during lethal peritonitis increased progressively to values 50- to 100-fold lower than the peak values observed after i.v. injections, and remained sustained until death. Whereas after i.v. challenge with 1000 LD50 of LPS, anti-TNF-alpha antibody fully protected mice from death and reduced serum IL-1 and IL-6 levels, anti-TNF-alpha antibody did not improve the survival of mice nor reduced serum IL-1 and IL-6 levels after i.p. bacterial challenge. In contrast to anti-TNF-alpha antibodies, anti-LPS antibodies were protective in the peritonitis model. Protection was accompanied by a striking reduction of bacterial numbers and of TNF-alpha, IL-1, and IL-6 levels in the serum, but the levels of these cytokines were only marginally affected in the peritoneal lavage fluid. This latter observation demonstrates that the local peritoneal cytokines did not diffuse readily into the circulation, thus suggesting that at least part of the circulating cytokines are produced systemically. In conclusion, the striking differences between cytokine profiles as well as the divergent efficacy of anti-TNF-alpha antibody after i.v. bolus and after i.p. challenges suggest that TNF-alpha may not be as important in the pathogenesis of lethal peritonitis than after lethal acute bacteremia.     

Serum and gastric fluid levels of cytokines and nitrates in gastric diseases infected with Helicobacter pylori

This case control study presents data on the concentrations of nitrite and nitrate and a variety of pro-inflammatory cytokines such as interleukin-1 beta (IL-1 beta), interleukin-2R (IL-2R), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor TNF-alpha in gastric fluid and serum. Patients with gastritis, gastric ulcer and gastric cancer are studied and grouped according to infection by Helicobacter pylori. The 208 patients who underwent upper gastrointestinal endoscopic examination were classified as follows; H. pylori-positive gastritis (n = 32), H. pylori-negative gastritis (n = 32), H. pylori-positive ulcers (n = 34), H. pylori-negative ulcers (n = 34), 43 patients with H. pylori-positive gastric cancer in addition to 33 H. pylori-negative healthy control individuals. Gastric fluids and blood samples were taken concomitantly. Cytokines and nitrite and nitrate determinations were attempted as soon as possible after collection of the samples. Nitrite and nitrate levels of serum and gastric fluids of H. pylori-positive gastritis and ulcers were higher than H. pylori-negative gastritis and ulcers. The concentrations of total nitrite and nitrate and cytokines (TNF-alpha, IL-2R, IL-6, and IL-8) in gastric fluids and sera of H. pylori-positive gastric cancer patients were higher than H. pylori-negative control groups. IL-1 beta level was significantly elevated in gastric fluid of infected cancer patients but not in serum. Taken together, the results suggest that an increase in cytokine-NO combination in gastric mucosa previously reported by many studies is not restricted to local infected gastric tissue but also detected in gastric fluid and sera of H. pylori-positive subjects and may have an important role in the pathogenesis and development of common gastric diseases.     

Immunoaffinity liquid chromatography-tandem mass spectrometry detection of nitrotyrosine in biological fluids: development of a clinically translatable biomarker

Measurement of nitrotyrosine levels in biological fluids can serve as a biomarker for oxidative/nitrative damage arising from formation of reactive nitrogen species, including peroxynitrite. Peroxynitrite is formed by the reaction of the superoxide radical (O2.-) with the nitric oxide radical (.NO) that is generated by nitric oxide synthase (NOS). This article describes an immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 3-nitrotyrosine at very low (picomolar) levels. Incorporation of a pronase digestion step prior to the immunoaffinity LC-MS/MS allowed for measuring not only free amino acid but also protein 3-nitrotyrosine in biological fluids. The use of an in-line antibody column allowed for increased specificity as compared with previously reported assays. The assay is linear over a range of 5 to 500 pg/ml (0.022-2.20 nM, r(2)=0.9987), with the lower detection limit being 5 pg/ml. In addition to its increased sensitivity and specificity, this assay showed great nitrotyrosine recovery from biological fluids when either nitrotyrosine or nitrotyrosine-containing peptides were added exogenously. The utility of this assay for nitrotyrosine as a clinically translatable biomarker was demonstrated by quantifying both free and total nitrotyrosine levels in various biological fluids, including urine, plasma, serum, cerebrospinal fluid (CSF), and synovial fluid (SF) from both preclinical species and human subjects. Thus, whether in an animal model of human disease or in a clinical setting, the quantification of nitrotyrosine levels should provide support for NOS-driven pathology and its blockade following therapeutic intervention.