Ex vivo cytokine production by whole blood cells from cancer patients.

It has been hypothesized that the development of cancer might partially result from a diminution of immunocompetence. Using ex vivo cytokine production by whole blood (WB) cells after polyclonal activation, we compared cytokine production levels of cancer patients to those of healthy controls. Seventeen patients without any prior treatment and attending the hospital for oncological surgery (for cancers of several origins) were enrolled in the study. WB was collected in heparinized tubes, diluted 1/10 in RPMI 1640 and incubated for 2, 4, 24, 48, and 72 h at 37 degrees C in the presence of 5 micrograms/ml PHA and 25 micrograms/ml LPS. Cytokine levels in the supernatant were measured by specific immunoassay kits. IL-10 levels after 24 h of culture, IFN-gamma and GM-CSF levels after 24 and 72 h of culture, and LIF levels after 72 h of culture were significantly lower in cancer patients than in healthy controls. No significant difference was observed for IL-1 beta, IL-2, IL-4, IL-8, and TNF-alpha production at any culture time. Our results suggest that the putative immunosuppression of cancer patients might be reflected by their reduced production of immunostimulated cytokines.

Adverse effect of abdominal operations on production of interferon-gamma.

OBJECTIVE: To assess the effects of abdominal operations on the production of cytokines as one of the mechanisms of postoperative immunosuppression. DESIGN: Prospective study. SETTING: University hospital, Belgium. SUBJECTS: 19 Selected patients who underwent operations for benign (n = 10) or malignant (n = 9) diseases. INTERVENTIONS: Whole blood was collected in heparinised tubes before operation and on postoperative days 1, 2, 3, 5, 7, and 9. After 1/10 dilution in culture medium the whole blood cells were stimulated with 5 micrograms/ml phytohaemagglutinin and 25 micrograms/ml lipopolysaccharide, and incubated at 37 degrees C in 5% carbon dioxide. Concentrations of interleukin 1 (IL-1), tumour necrosis factor alpha (TNF alpha), and interleukin 6 (IL-6) were measured at 24 hours, and interferon-gamma and interleukin 2 (IL-2) were measured at 72 hours, with commercially available assays. OUTCOME MEASURES: Production of the monokines IL-1, TNF alpha, and IL-6, and of the lymphokines IL-2 and interferon-gamma, postoperatively. The monokines were expressed as a percentage of the preoperative values/monocyte, and the lymphokines as a percentage of preoperative values/lymphocyte. RESULTS: Production of IL-1 and TNF alpha, but not IL-6, decreased immediately after operation then returned to preoperative values. Production of IL-2 and interferon-gamma were significantly reduced immediately after operation, and that of interferon-gamma was still depressed on the ninth postoperative day. CONCLUSION: Cytokine production is altered after abdominal operations. The production of interferon-gamma may be a more sensitive indicator of altered immune response and vulnerability to infections and tumour growth than concentrations of other cytokines.

Characterisation of monoclonal antibodies against human interleukin-10 and their use in an ELISA for the measurement of this cytokine.

Biological and biochemical characteristics of monoclonal antibodies (MABs) raised against human interleukin-10 (IL-10) are described as well as their use in the design of a specific ELISA for the measurement of the cytokine. 21 murine anti-human interleukin-10 (IL-10) MABs were obtained by fusion of splenocytes from mice immunized against human recombinant IL-10 with SP2/0 myelomatous cells. These antibodies define three major antigenic areas on the IL-10 molecule, one of which comprises epitopes involved in receptor binding and induction of biological activity. They recognize recombinant human IL-10 with affinities ranging from 1.3 x 10(-7) to 3 x 10(-11), as well as natural IL-10. Most of them also recognize viral IL-10 (vIL-10) encoded by the Epstein-Barr virus (EBV). A specific human-IL-10 ELISA has been developed using two MABs (18 and 19) as capture antibody and one MAB (17) as detector. The sensitivity (3 pg/ml), precision (intra-assays < 4%), reproducibility (interassay < 3%), and accuracy (recoveries, ranging between 84 and 107%, in several fluids) of the assay, plus its excellent performance in dilution tests, and the lack of interference when in the presence of possible cross-reactive substances, permits accurate cytokine measurement in biological fluids such as serum, plasma, bronchoalveolar lavage, urine and culture supernatants. Using the assay, IL-10 was measurable in the plasma of patients with septic shock (range 11-2740 pg/ml) whereas IL-10 plasma levels were < 7.8 pg/ml in healthy volunteers.

Cytokines and organ transplantation. A review.

Cytokines regulate both aspecific inflammatory responses and specific immune responses. Inflammatory changes occur in the organ transplant as a result of tissue trauma and ischemia/reperfusion in the organ donor and at the time of transplant operation. There is a possibility that cytokines play a role in mediating theses changes. These aspecific inflammatory changes may not only affect graft function but also influence graft immunogenicity (enhanced MHC and adhesion molecule expression) and thus, vulnerability to rejection. Cytokines orchestrate the specific immune response elicited by organ transplantation. Relevance of cytokines to the rejection reaction is multifactorial in nature: 1) promotion of the proliferation an differentiation of specific alloreactive T and B cells clones and differentiation and activation of CTL and NK cells, 2) chemotactic effect and induction of the expression of adhesion molecules, 3) enhancement of MHC class I and II expression, and 4) direct cytotoxic effect on the target grafted cells. Therefore, modulation of cytokine activity either specifically (monoclonal antibody, soluble receptor, etc.) or aspecifically (cyclosporin, FK 506, Rapamycin, steroids, etc.) is essential in controlling graft rejection. Determination of circulating cytokines and cytokines measurement within the biological fluids produced by an organ transplant may help in the diagnosis of rejection episodes and other complications following organ transplantation.

An ELISA for the measurement of human leukemia inhibitory factor in biological fluids and culture supernatants.

A new monoclonal antibody-based ELISA for leukaemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) measurements is described. The sensitivity (56 pg/ml after 4 h incubation, 14 pg/ml after 24 h incubation), precision (intra-assays < 5%), reproducibility (interassay < 10%), and accuracy (recoveries, ranging between 98 and 119%, in several fluids) of the assay, plus its excellent performance in dilution tests, and the lack of interference when in the presence of possible cross-reactive substances guarantee accurate cytokine measurement in biological fluids such as serum, plasma, synovial fluid, follicular fluid, urine and culture supernatants. Using the assay, LIF/HILDA was measurable in supernatants after in vitro whole blood stimulation with phytohemagglutinin (PHA), OKT3, and phorbol myristate acetate (PMA) but not with lipopolysaccharide (LPS) or Ca ionophore. LIF/HILDA production was not measurable until after 24 h of culture, when cytokine levels were seen to increase linearly in the supernatant to reach values of up to 40 ng/ml after 96 h of culture. Finally, a good correlation was found (r = 0.96; p < 0.0001; y = 23.1x + 233) between the LIF/HILDA values obtained using the ELISA and DA-1a bioassay.

Novel method for the measurement of cytokine production by a one-stage procedure.

A new one-step culture-immunoassay procedure is described for testing cytokine production by immunocompetent cells in whole blood (WB) without the need for an isolation step. Briefly, WB samples or distilled water were added to RPMI medium containing specific anti-cytokine peroxidase-labelled monoclonal antibodies and incubated in micro-well plates coated with specific capture monoclonal antibodies, directed against distinct epitopes of the cytokine, and containing dried polyclonal activators (5.625 micrograms LPS + 1.125 micrograms PHA) or dried standards respectively. The optimalisation of the assay is described for an extended measurement range. The best compromise between sensitivity and linearity was obtained with the addition of 50 ng/well for TNF-alpha and IL-6 or 100 ng/well for IFN-gamma of unconjugate antibodies to the corresponding conjugate. The kinetics of individual production of each cytokine in WB of normal healthy donors showed values entering the standard range following incubation times of between 2 and 8 h for TNF-alpha, 2 and 4 h for IL-6, and 4 and 24 h for IFN-gamma. The sensitivity, the precision (intra-assay CVs) and the reproducibility (interassay CVs) of the assays were as follows: 70 pg/ml, < or = 14% and < or = 11% for TNF-alpha; 25 pg/ml, < or = 11% and < or = 16% for IL-6; 25 pg/ml, < or = 19% and < or = 20% for IFN-gamma. The accuracy (% of recovery) of the assays was in the order of 100% and between 40 and 60% in the absence or presence of polyclonal activators, reflecting the occurrence of an active production/consumption mechanism during the activation.

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.

Intraoperative cytokines production during orthotopic liver transplantation.

In summary, we established that a significant production of the monokines interleukin-6, tumor necrosis factor apha, and interleukin-1 occurred during orthotopic liver transplantation whereas the lymphokines interferon gamma and interleukin-2 were not detected. Levels of interleukin-6 reached their maximum values before and especially at the end of the anhepatic phase. They remained high after the anhepatic phase, i. e. after reperfusion of the new livers. Tumor necrosis factor alpha and interleukin-1 reached their maximum values after the anhepatic phase. Not only were interleukin-6, tumor necrosis factor alpha, and interleukin-1 present in the serum but they could also be detected in the bile produced by these new livers. Mechanisms of monokine production during orthotopic liver transplantation is multifactorial in origin and further studies will have to evaluate the relative contribution of the various factors involved. The possibility of an association between peroperative monokines and transplant outcome and their potential clinical implication will have to be elucidated.

Production of interferon gamma by peripheral blood mononuclear cells from normal subjects and from patients with rheumatoid arthritis.

A radioimmunoassay for human interferon gamma (IFN-gamma) has been carried out using a recombinant glycosylated interferon (Hu IFN-gamma) as tracer, the N.I.H. reference preparation (Gg 23-901-530) and a polyclonal rabbit antiserum. The assay is highly specific for IFN-gamma: there is no cross-reaction either with interferons alpha and beta, Interleukins 1 and 2, tumor necrosis factor alpha and beta or with various brain peptides. The sequential saturation procedure allowed a sensitivity of 0.4 U/ml with intra and between assay coefficients of variation less than 8 and 12%, respectively. The in-vitro production of IFN-gamma by peripheral blood mononuclear cells (P.B.M.C.) was also measured. In unstimulated cultures, IFN-gamma production remained undetectable, i.e. below the 0.4 U/ml sensitivity level. After stimulation of P.B.M.C. from normal subjects with increasing amounts of PHA, both the 3H-thymidine incorporation and IFN-gamma release followed bell-shaped curves. There was no significant difference of 3H-thymidine incorporation between PHA stimulated cultures (0.2 and 2.5 ug/ml) from normal subjects (36 cases) and those with active (16 cases) or non-active (14 cases) rheumatoid arthritis. At two PHA concentrations of 0.2 and 2.5 ug/ml, mononuclear cells from patients with active disease produced significantly less IFN-gamma than those from either controls or cases with non-active disease.