Lysophospholipid enhancement of human T cell sensitivity to diphtheria toxin by increased expression of heparin-binding epidermal growth factor.

The effects of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) on T cell expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), the diphtheria toxin (DT) receptor, were investigated in the Tsup-1 cultured line of human CD4+ 8+ 3low T lymphoblastoma cells. Tsup-1 cells bear endothelial differentiation gene (edg)-2 and -4-encoded G protein-coupled receptors (GPCRs) for LPA and Edg-3 and -5 GPCRs for S1P. Suppression by DT of Tsup-1 cell protein synthesis was enhanced by LPA and S1P, with lipid structural specificity similar to that required for their recognition by Edg receptors. LPA and S1P increased the Tsup-1 cell level of immunoreactive HB-EGF, and neutralizing antibodies to HB-EGF inhibited LPA and S1P enhancement of Tsup-1 cell susceptibility to DT. Stabilized transfection of Tsup-1 cells with a combination of plasmids encoding Edg-2 plus -4 antisense mRNA suppressed the levels of Edg-2 and -4, but not Edg-3 and -5, in Western blots and reduced in parallel the increments in HB-EGF and susceptibility to DT evoked by LPA but not S1P. Similar transfection with Edg-3 plus -5 antisense plasmids suppressed Tsup-1 cell levels of immunoreactive Edg-3 and -5, but not Edg-2 or -4, and concurrently reduced S1P-, but not LPA-, induced Tsup-1 cell increases in both HB-EGF and susceptibility to DT. Edg GPCR-mediated LPA and S1P enhancement of T cell sensitivity to DT, thus, may be attributable to increased expression of the DT receptor HB-EGF.

Chemokine production and adhesion molecule expression by neural cells exposed to IL-1, TNF alpha and interferon gamma.

We investigated the effect of TNF alpha, IL-1alpha and IFN gamma on two neuroblastoma (NB) cell lines (SK-N-SH and SK-N-MC). These lines responded differentially to IL-1alpha, TNF alpha and IFN gamma for MCP-1 and IL-8 production and expression of the ICAM-1 and VCAM-1 adhesion molecules. None of the cytokines induced MCP-1 or IL-8 on SK-N-MC cells. Both chemokines were produced in response to IL-1alpha by SK-N-SH cells, while TNF alpha induced mainly MCP-1 production. Addition of IFN gamma decreased IL-8, but not MCP-1 production. These responses correlated with monocyte and neutrophil chemotactic activity in NB culture supernatants. This activity was neutralized by antibodies to IL-8 and MCP-1. The expression of ICAM-1 on SK-N-MC was up-regulated by TNF alpha or IFN gamma, while IL-1alpha also upregulated ICAM-1 on SK-N-SH cells. VCAM-1 expression on SK-N-SH was induced by IL-1alpha and TNF alpha and IFN gamma synergized with TNF alpha in this respect on both NB cell lines. These results suggest that mechanisms for chemokine production and VCAM-1 and ICAM-1 upregulation by inflammatory cytokines differ and IFN gamma, in conjunction with TNF alpha, stimulate neural cell responses (high MCP-1 and VCAM-1 and decreased IL-8) favouring mononuclear cell recruitment.

Antioxidants inhibit the in vitro production of inflammatory cytokines in Crohn's disease and ulcerative colitis.

BACKGROUND: Modulation of cytokine secretion may be of interest in the treatment of Crohn's disease or ulcerative colitis. METHODS: The effect of three antioxidants - butylated hydroxyanisol, tetrahydropapaveroline and nordihydroguaiaretic acid - on the production of tumour necrosis factor (TNF), interleukin (IL) 1, IL-6 and IL-8 (measured by enzyme-linked immunosorbent assay) by peripheral mononuclear cells and biopsies of inflamed colonic mucosa from inflammatory bowel disease patients were studied. RESULTS: We observed a decrease in IL-1 and IL-6 production by peripheral mononuclear cells from inflammatory bowel disease patients (approximately 50% of control). The three drugs did not decrease IL-6 and IL-8 secretion by colonic biopsies, whereas they did inhibit IL-1 and, to some degree, TNF production. The cytokine-inhibitory effect of antioxidants seems to be more pronounced in ulcerative colitis than in Crohn's disease. CONCLUSION: Our results suggest that the studied antioxidants, or related compounds, may be of interest in inflammatory bowel disease treatment.

In vitro effects of oxpentifylline on inflammatory cytokine release in patients with inflammatory bowel disease.

BACKGROUND: Inflammatory cytokines, including tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 beta, have been implicated as primary mediators of intestinal inflammation in inflammatory bowel disease. AIM: To investigate the in vitro effects of oxpentifylline (pentoxifylline; PTX; a phosphodiesterase inhibitor) on inflammatory cytokine production (1) by peripheral mononuclear cells (PBMCs) and (2) by inflamed intestinal mucosa cultures from patients with Crohn's disease and patients with ulcerative colitis. METHODS: PBMCs and mucosal biopsy specimens were cultured for 24 hours in the absence or presence of PTX (up to 100 micrograms/ml), and the secretion of TNF-alpha, IL-1 beta, IL-6, and IL-8 determined by enzyme linked immunosorbent assays (ELISAs). RESULTS: PTX inhibited the release of TNF-alpha by PBMCs from patients with inflammatory bowel disease and the secretion of TNF-alpha and IL-1 beta by organ cultures of inflamed mucosa from the same patients. Secretion of TNF-alpha by PBMCs was inhibited by about 50% at a PTX concentration of 25 micrograms/ml (IC50). PTX was equally potent in cultures from controls, patients with Crohn's disease, and those with ulcerative colitis. The concentrations of IL-6 and IL-8 were not significantly modified in PBMCs, but IL-6 increased slightly in organ culture supernatants. CONCLUSIONS: PTX or more potent related compounds may represent a new family of cytokine inhibitors, potentially interesting for treatment of inflammatory bowel disease.

Transforming growth factor-beta 1 inhibits synthesis of cytokines in endotoxin-stimulated human whole blood.

OBJECTIVE: To determine the potency of transforming growth factor-beta (TGF-beta) for inhibiting proinflammatory cytokine synthesis in endotoxin-stimulated human whole blood. DESIGN: Endotoxin-stimulated whole blood from healthy volunteers as an ex vivo model of endotoxemia was incubated with different concentrations of TGF-beta 1. Cytokine levels in plasma with a bioassay (for tumor necrosis factor alpha) or an enzyme-linked immunosorbent assay (for interleukin [IL]-1 beta and IL-6), messenger RNA (mRNA) expression with northern blotting, and protein levels with Western blotting were determined. RESULTS: High TGF-beta 1 concentrations (> 100 pg/mL) inhibited (P < .05) secretion of tumor necrosis factor alpha, IL-1 beta, and IL-6 into lipopolysaccharide-stimulated whole blood, while low concentrations (< 50 pg/mL) were ineffective. Moreover, TGF-beta 1 inhibited mRNA expression of tumor necrosis factor alpha and IL-6 in a dose-dependent manner. In contrast, neither IL-1 beta mRNA expression nor IL-1 beta protein synthesis were attenuated by TGF-beta 1. CONCLUSION: Transforming growth factor-beta 1, with its downregulatory effect on the synthesis and release of proinflammatory cytokines by phagocytic cells, represents an inhibitor of endotoxin-induced inflammatory reactions.

Increased production of tumour necrosis factor-alpha interleukin-1 beta, and interleukin-6 by morphologically normal intestinal biopsies from patients with Crohn's disease.

BACKGROUND: Increasing evidence points to a important role for inflammatory cytokines for the pathogenesis of Crohn's disease. AIM: To compare the secretion rate of tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) by morphologically normal and inflamed intestinal mucosa from patients with Crohn's disease. RESULTS: Organ cultures of intestinal biopsy specimens taken from areas of affected mucosa from patients with Crohn's disease spontaneously produced increased amounts of TNF-alpha, IL-1 beta, and IL-6 compared with controls but also biopsy specimens taken in macroscopically and microscopically unaffected areas in the same patients. Concentrations of IL-1 beta and IL-6 measured in the supernatant fluid of biopsy cultures were positively correlated with the degree of tissue involvement measured by both endoscopic and histological grading. By contrast, TNF-alpha concentrations were not correlated to endoscopic and histological grading. CONCLUSIONS: These consistently raised TNF-alpha, IL-1 beta and IL-6 secretions by normal appearing mucosa from patients with Crohn's disease provide evidence for a sustained immune stimulation in Crohn's disease even in the absence of patent inflammation. The results shed a new light on the role of inflammatory cytokines in the onset of intestinal tissue damage in Crohn's disease and suggest that the range of intestinal lesions in Crohn's disease may be wider than suspected on the basis of regular endoscopic and histological examinations.

A human tumor necrosis factor p75 receptor agonist stimulates in vitro T cell proliferation but does not produce inflammation or shock in the baboon.

Tumor necrosis factor (TNF) is a potentially useful adjunct to anticancer therapies. However, the clinical utility of TNF has been limited by generalized toxicity and hypotension. Recently, studies have begun to dissect the individual proinflammatory and immunologic responses that result from TNF binding to its two cellular receptors, p55 and p75, in an attempt to develop TNF receptor agonists with reduced systemic toxicity. To evaluate a p75 receptor selective TNF mutant (p75TNF), TNF and p75TNF were administered to healthy anesthetized baboons. Intravenous infusion of the p75TNF produced none of the hemodynamic changes seen after the infusion of TNF. Infusion of p75TNF also failed to induce the plasma appearance of interleukins 6 and 8. However, p75TNF enhanced in vitro baboon thymocyte proliferation to concanavalin A, and infusion of p75TNF resulted in increased soluble p55 and p75 receptor plasma concentrations. Local skin necrosis and tissue neutrophil infiltration were seen after subcutaneous injections of TNF and p55TNF. Subcutaneous injection of p75TNF did not result in skin necrosis but did result in a modest dermal infiltration of lymphocytes and macrophages. The findings suggest that p75TNF may stimulate T cell proliferation without the systemic and local toxicity seen with TNF.

Release of anti-inflammatory mediators after mechanical trauma correlates with severity of injury and clinical outcome.

Excessive synthesis of proinflammatory cytokines [tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta] after trauma has been correlated with poor outcome. Recently, naturally occurring inhibitors of TNF-alpha and IL-1 beta have been characterized such as soluble TNF receptors (sTNFRs) and IL-1 receptor antagonist (IL-1ra). The present study was undertaken to determine whether injury results in a rise of circulating sTNFRs and IL-1ra. If so, whether plasma levels of these anti-inflammatory mediators correlate with severity of injury and clinical outcome of these patients. Injured patients (n = 213) showed significantly increased sTNFR and IL-1ra plasma levels throughout the observation period of 14 days, compared with healthy volunteers (n = 127). Patients with severe injury (Injury Severity Score > 16 points) revealed higher levels (p < 0.05) of sTNFRs and IL-1ra than patients with minor trauma (Injury Severity Score < or = 16 points). Patients who died from injury demonstrated increased (p < 0.05) sTNFR p55 and IL-1ra plasma levels, compared with survivors. Thus, anti-inflammatory mechanisms are activated after trauma dependent on severity of injury. Because increased plasma levels of anti-inflammatory reacting proteins portended poorly for patient survival, these mediators may contribute to prediction of outcome after severe injury.

Production of interleukin-6 in human osteoblasts and human bone marrow stromal cells: evidence that induction by interleukin-1 and tumor necrosis factor-alpha is not regulated by ovarian steroids.

Studies in murine models of osteoporosis have suggested the hypothesis that ovarian steroids may control osteoclastic bone remodeling by limiting the production of interleukin-6 (IL-6) from osteoblasts and bone marrow stromal cells. To investigate this hypothesis in a human model, we have examined 12 separate strains of normal human osteoblasts (HOB) and 11 separate strains of human bone marrow stromal cells (HBMSC) and determined whether ovarian steroids regulate the induction of IL-6 by interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) or IL-1 + TNF. Treatment with IL-1, TNF or IL-1 + TNF resulted in the induction of IL-6 from both cell types with IL-1 + TNF inducing a synergistic induction of IL-6 in HOB (24- to 324-fold) and HBMSC (35-288 fold). Addition of 17 beta-estradiol or progesterone did not significantly alter IL-6 messenger RNA or protein levels in either HOB or HBMSC cultures stimulated with IL-1, TNF or IL-1 + TNF. Cultures incubated up to 96 h with the steroids did not affect IL-6 expression. Furthermore ovarian steroids did not affect IL-6 production in either HBMSC cultures representative of preosteoblasts or HOB cultures representative of highly differentiated osteoblasts. Specific chloramphenicol acetyl transferase assays and reverse transcriptase-polymerase chain reaction studies also demonstrated that the lack of an estrogen effect was not due to the failure of HOB to express functional estrogen receptors. Therefore, we conclude that the regulation of human osteoclastic bone remodeling by ovarian steroids does not occur through the direct regulation of IL-6 gene transcription or protein secretion in either early stages of osteoblast differentiation or the differentiated osteoblast.

Production of monoclonal antibodies using a secretion capture report web.

We describe a novel method for the production of monoclonal antibodies using a secretion capture report web (SCRW). Following HAT selection in bulk culture, individual hybridomas are encapsulated in biotinylated agarose drops. Antibody secreted by the hybridoma is captured within the agarose drop using an avidin bridge and biotinylated anti-mouse immunoglobulin. The secreted antibody is detected by a fluorescent reporter which can be either a second anti-mouse antibody or an antigen. The binding of the reporter can be quantitated and the desired hybridoma directly cloned by flow cytometry. Multiparameter (i.e., two-color) reporter analysis can also be used to selectively enrich and clone rare hybridomas secreting antibodies directed to unique epitopes. The method allows the characterization of thousands of clones per second and the isolation of hundreds of clones per day.

PEG-BP-30 monotherapy attenuates the cytokine-mediated inflammatory cascade in baboon Escherichia coli septic shock.

Septic shock following gram-negative infection is a leading cause of mortality in critically ill patients, accounting for nearly 200,000 deaths a year. The exaggerated production of tumor necrosis factor-alpha (TNF alpha) is known to contribute to hemodynamic collapse and the hematological dyscrasia associated with gram-negative sepsis. Although previous studies have shown TNF alpha antibodies and TNF immunoadhesins to be effective in experimental gram-negative sepsis, we postulated that administration of a novel construct of two modified soluble p55 receptors linked to polyethylene glycol (PEG-BP-30) would also attenuate the hemodynamic and hematologic alterations to lethal Escherichia coli septic shock in non-human primates. Nine adult female and male baboons (Papio anubis), weighing 10-17 kg, were anesthetized and invasively monitored. The nine animals were randomized to receive either 0.2 mg/kg body wt PEG-BP-30 (n = 3), 5.0 mg/kg body wt PEG-BP-30 (n = 3), or placebo (n = 3). One hour after pretreatment, animals were infused with 5-10 x 10(10) CFU/kg of live E. coli iv and vital signs were recorded for the next 8 hr. Arterial blood was drawn for baseline parameters and throughout the study to obtain total and differential white blood cell and platelet counts and cytokine levels (TNF alpha, IL-1 beta, IL-6, IL-8). E. coli bacteremic baboons receiving only placebo demonstrated a significant fall in mean blood pressure and leukopenia. Two of the three animals expired. In contrast, five of the six baboons receiving the PEG-BP-30 survived and these animals exhibited markedly attenuated declines in blood pressure and leukocyte numbers.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion capture and report web: use of affinity derivatized agarose microdroplets for the selection of hybridoma cells.

A novel assay is described which allows the entrapment and detection of the immunoglobulin secreted from individual viable hybridoma cells using a secretion capture and reporter web (SCRW). By encapsulating the cells in agarose microdroplets which have been derivatized to create a fluorescent antigen-specific sandwich assay, flow cytometry can be used to identify and sort productive cells from a heterogeneous population. Using agarase, the cells can be recovered from the microdroplets and clonally expanded after selection. The assay has been used to reclone rare secretors from hybridoma cultures and to enhance the production of cultures with poor producers. The assay is easily generalized for the detection of any secreted protein for which specific antibodies or other ligands are available.

Synthesis of interleukin-1 alpha, interleukin-6, and interleukin-8 by cultured human nasal epithelial cells.

Nasal epithelium forms the initial barrier between the environment and the respiratory system and may be a potential source of proinflammatory interleukins, which contribute to the pathophysiology of allergic and nonallergic rhinitis. To explore this possibility, epithelium and cultured human nasal epithelial cells from nasal turbinates of patients undergoing surgery for treatment of upper airway obstruction were examined for the spontaneous expression of interleukin (IL)-1 alpha, IL-1 beta, IL-6, and IL-8. Human nasal epithelial cell lysates and culture supernatants were assayed by two-site ELISAs specific for IL-1 alpha, IL-1 beta, IL-6, or IL-8. Maximum concentrations of these cytokines in supernatants ranged from approximately 0.2 to 2 ng/ml for IL-1 alpha, 1.5 to 7 ng/ml for IL-6, and 100 to 3000 ng/ml for IL-8. IL-1 alpha was predominantly cell-associated, whereas most of the IL-8 and all of the IL-6 were detected in the supernatant. Little or no IL-1 beta was detected by ELISA in the supernatants or cell lysates. Whole tissue turbinates and isolated epithelium were also examined for IL-1 beta, IL-6, and IL-8 mRNA expression by Northern blot analysis. IL-6 and IL-8 mRNAs were detected, whereas IL-1 beta mRNA was not. Furthermore, IL-6 and IL-8 release from human nasal epithelial cell cultures was enhanced by addition to the cultures of lipopolysaccharide, and IL-6 release was inhibited by polymyxin B. Thus human nasal epithelium may be a major source of IL-1 alpha, IL-6, and IL-8 in allergic and nonallergic rhinitis. Production of those proinflammatory cytokines by epithelial cells of the nasal and sinus mucosa may contribute to the pathologic and clinical events that occur in these diseases.

HIV-1 infection of macrophages promotes long-term survival and sustained release of interleukins 1 alpha and 6.

HIV infection of macrophages in vivo may result in activation of monokine genes and cause persistent release of immunomodulatory and inflammatory cytokines. Studies that have examined cytokine (IL-1, IL-6, and TNF-alpha) activation by in vitro infection of normal peripheral blood mononuclear cells (PBMCs) with HIV-1 have produced conflicting results. The present study shows that for monokine induction by HIV-1-IIIB preparations derived from the H9 tumor cell line, partial purification of virus particles is essential. Infectious HIV-1 induces the release of high levels of IL-1 alpha, IL-1 beta, and IL-6 bioactivity by adherent PBMCs in the first 3 days following in vitro infection, but only IL-1 alpha and IL-6 continue to be released over several weeks of culture. High levels of bioactive IL-1 beta were released only up to 72 hr following infection, although intracellular IL-1 beta was detectable for at least 3 weeks. No TNF-alpha bioactivity or immunoreactive protein was detectable at > 48 hr in HIV-infected cultures. This time course of monokine release was dependent on the number of infectious particles added to PBMC cultures. In long-term cultures (> 1 month) HIV infection was found to promote the viability of macrophages. The finding of sustained release of IL-1 alpha and IL-6 by infected macrophages, without additional stimulation, suggests that these mediators are released by HIV-1-infected macrophages in AIDS patients, where they may interfere with proper immune regulation.

The diagnostic and prognostic value of amniotic fluid white blood cell count, glucose, interleukin-6, and gram stain in patients with preterm labor and intact membranes.

OBJECTIVE: Our goal was to compare the value of amniotic fluid tests in the detection of microbial invasion of the amniotic cavity and in the relationship with the amniocentesis-to-delivery interval and neonatal complications in patients with preterm labor and intact membranes. STUDY DESIGN: Amniotic fluid was retrieved by transabdominal amniocentesis from 120 patients with preterm labor and intact membranes. Fluid was cultured for aerobic and anaerobic bacteria and for mycoplasmas. Amniotic fluid analysis included a Gram stain, white blood cell count, glucose and interleukin-6 determinations. Logistic regression and Cox's proportional hazards model were used for analysis. RESULTS: (1) The prevalence of positive amniotic fluid cultures was 9.2% (11/120); (2) patients with microbial invasion had a shorter amniocentesis-to-delivery interval and a higher neonatal complications rate than patients with a negative culture; (3) the most sensitive test for the detection of microbial invasion of the amniotic cavity was amniotic fluid interleukin-6 determinations (cutoff 11.3 ng/ml) (sensitivity; for interleukin-6 100%, for glucose 81.8%, for white blood cell count 63.6%, and for Gram stain 63.6%; p < 0.05 for all comparisons); (4) the most specific test was the Gram stain of amniotic fluid (specificity: for Gram stain 99.1%, for white blood cell count 94.5%, for interleukin-6 82.6%, and for glucose 81.6%; p < 0.01 for all); (5) of all amniotic fluid tests, interleukin-6 determinations were the only ones that had significant relationship with the amniocentesis-to-delivery interval and neonatal complications. CONCLUSION: Interleukin-6 concentrations in amniotic fluid are better indicators of microbial invasion of the amniotic cavity, amniocentesis-to-delivery interval, and neonatal complications than the amniotic fluid Gram stain, glucose concentration, or white blood cell count.

A comparative study of the diagnostic performance of amniotic fluid glucose, white blood cell count, interleukin-6, and gram stain in the detection of microbial invasion in patients with preterm premature rupture of membranes.

OBJECTIVE: Our aim was to compare the value of amniotic fluid tests for the detection of microbial invasion of the amniotic cavity and in the prediction of the amniocentesis-to-delivery interval and neonatal complications in patients with preterm premature rupture of membranes. STUDY DESIGN: Amniotic fluid was obtained by transabdominal amniocentesis from 110 consecutive patients with preterm premature rupture of membranes. Fluid was cultured for aerobic and anaerobic bacteria, as well as mycoplasmas. Amniotic fluid analysis included a Gram stain examination, white blood cell count, and glucose and interleukin-6 determinations. Logistic regression and survival techniques (proportional hazards model) were used for statistical analysis. RESULTS: (1) The prevalence of positive amniotic fluid cultures in patients with preterm premature rupture of membranes was 38% (42/110); (2) patients with microbial invasion had a shorter amniocentesis-to-delivery interval and a higher neonatal complication rate than patients with negative cultures; (3) the most sensitive test for the detection of microbial invasion of the amniotic cavity was amniotic fluid interleukin-6 determinations (cutoff 7.9 ng/ml) (sensitivity: for IL-6 80.9%; for white blood cell count 57.1%; for glucose 57.1%; for Gram stain 23.8%; p < 0.05 for all comparisons); (4) the most specific test for the detection of microbial invasion was the Gram stain of amniotic fluid (specificity: for Gram stain 98.5%; for white blood cell count 77.9%; for interleukin-6 75%; for glucose 73.5%; p < 0.01 for all); (5) of all amniotic fluid tests, interleukin-6 determination was the only test that had significant clinical value in the prediction of the amniocentesis-to-delivery interval and neonatal complications. CONCLUSION: Interleukin-6 concentrations in amniotic fluid are a better predictor of microbial invasion of the amniotic cavity, amniocentesis-to-delivery interval and neonatal complications than the amniotic fluid Gram stain, glucose, or white blood cell count in patients with preterm premature rupture of membranes.

Antibodies chaperone circulating IL-6. Paradoxical effects of anti-IL-6 "neutralizing" antibodies in vivo.

In the baboon or the mouse, a stimulus such as LPS, TNF, or IL-1 typically led to a rapid induction of circulating IL-6, the levels peaked by 2 to 3 h and then declined to near-baseline values by 6 to 8 h. Administration to baboons or mice of "neutralizing" anti-IL-6 mAb followed by an IL-6 inducer led to a marked and sustained increase in circulating IL-6 levels. IL-6 Ag, IL-6 biologic activity, neutralizing anti-IL-6 mAb, and IL-6/anti-IL-6-mAb complexes could all be observed for an extended period of time (beyond 8 h) in the circulation of such animals. Nevertheless, in mice, if the anti-IL-6 mAb had been administered before the IL-6 inducer, there was a reduction in the in vivo IL-6-induced stimulation of fibrinogen levels, indicating that most of the intravascular IL-6 was not readily available for eliciting hepatocyte effects under these experimental conditions. Intraperitoneal administration into mice of mixtures of murine rIL-6 or human rIL-6 together with their respective anti-IL-6 mAb led to a marked increase in the appearance and longevity in the peripheral circulation of the exogenously administered murine or human rIL-6 species in a biologically active form. Varying the ratio of human rIL-6 to anti-human IL-6 mAb indicated that a molar ratio of 1:1 was sufficient for the ability of mAb to chaperone IL-6 in the murine circulation. Human rIL-6 mixed with "neutralizing" mAb in the approximate ratio 1:1 elicited an enhanced fibrinogen response in vivo in the mouse; an IL-6:mAb ratio of 1:125 led to a reduction in the fibrinogen response even though the levels of circulating B9 bioactivity and of human rIL-6-Ag were maximal under these conditions. Gel-filtration chromatographic and Western blotting analyses of IL-6 present in vivo in the mAb-free baboon revealed that although the IL-6 Ag was largely present in high molecular mass complexes of size 400 kDa in association with soluble IL-6 receptor, the B9 bioactivity was largely of low molecular mass (20 kDa). In contrast, in the anti-IL-6 mAb-treated baboon or mouse, the IL-6 Ag and bioactivity were both largely in complexes of 200 kDa. Thus, the binding of IL-6 in the intravascular compartment to other proteins, anti-IL-6 mAb in the present studies, gives IL-6 unexpected biochemical and pharmacologic properties in vivo.

Amniotic fluid interleukin-6 determinations are of diagnostic and prognostic value in preterm labor.

PROBLEM: The purpose of this study was to determine if amniotic fluid concentrations of the interleukin-6 (IL-6) are of value in diagnosis of microbial invasion of the amniotic cavity and in the prediction of failure of tocolysis, preterm delivery and perinatal morbidity and mortality. METHOD: Amniotic fluid was obtained by transabdominal amniocentesis from 146 consecutive patients admitted with the diagnosis of preterm labor and intact membranes. Fluid was cultured for aerobic and anaerobic bacteria as well as for mycoplasmas. Amniotic fluid IL-6 levels were measured using a monoclonal antibody-based enzyme-linked immunosorbent assay with a sensitivity of 0.03 ng/ml. Logistic regression and Cox's proportional hazards model were used to examine the effect of several variables on dichotomous outcomes or interval to delivery. RESULTS: Patients with a positive amniotic fluid culture had a significantly higher amniotic fluid IL-6 concentrations than patients with a negative culture (median 91.2 ng/ml, range 0.9 to 437 ng/ml versus median 0.4 ng/ml, range < 0.3 to 195 ng/ml, respectively; P < .0001). An amniotic fluid IL-6 concentration of greater than or equal to 11.3 ng/ml had a sensitivity of 93.3% (14 of 15) and a specificity of 91.6% (120 of 131). All patients with an amniotic fluid IL-6 concentration above 11.3 ng/ml and a negative amniotic fluid culture (N = 11) delivered preterm and all placenta available for examination (N = 7) had histologic evidence of chorioamnionitis. Amniotic fluid concentrations of IL-6 were an independent predictor of preterm delivery, amniocentesis-to-delivery interval and neonatal morbidity and mortality. Moreover, IL-6 concentrations added significant information to the prediction of these outcomes to that provided only by clinical information such as cervical dilatation, gestational age at admission or at delivery. CONCLUSION: IL-6 is a sensitive and rapid test for the detection of microbial invasion of the amniotic cavity and for identifying women at risk for spontaneous preterm delivery and neonates at risk for morbidity and mortality.