Establishment of strongly neutralizing monoclonal antibody to human interleukin-6 and its epitope analysis

doi:10.1016/S0006-291X(89)80027-0

Biochemical and Biophysical Research Communications

Volume 165, Issue 2, 15 December 1989, Pages 728-734

https://doi.org/10.1016/S0006-291X(89)80027-0 Get rights and content

Summary

Three monoclonal antibodies against human interleukin-6 were established and characterized. One antibody was shown to strongly neutralize both the Ig-inducing and hybridoma/plasmacytoma growth activity of interleukin-6. The results of its epitope analysis using protease treated interleukin-6 and immobilized antibody indicated that this neutralizing antibody binds to a peptide corresponding to Leu151-Lyauthor71 of interleukin-6 molecule. Further analysis using synthetic peptides showed that a shorter peptide corresponding to Ala153-Thr162 can also inhibit the binding of the antibody to interleukin-6. These results suggest that this carboxyl-terminal region plays a crucial role in interleukin-6 functions.

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One aliquot was ready for determination of fasting blood glucose, blood urea, serum creatinine, lipid profile, serum albumin and total protein using conventional methods of clinical chemistry. The other aliquots were stored at −70 °C for measurement of serum visfatin by an enzyme-linked immunosorbent assay (Human visfatin ELISA kit, DRG International Inc., USA) [14], circulating adhesion molecules; intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), using commercially available enzyme-linked immunosorbent assay (Quantikine human sICAM-1 & sVCAM-1, R & D Systems, Europe Ltd, Abington, UK) [15], C-reactive protein using a high-sensitivity latex-enhanced immunonephelometric assay (Dade Behring, Germany) [16] and interleukin-6 using a commercially available double-sandwich enzyme-linked immunosorbent assay (Quantikine, R & D Systems, Minneapolis, Minn) [17]. After fasting overnight, each subject rested quietly in a supine position for 15 min, and baseline vital signs were recorded.
Endothelial dysfunction (ED) is closely linked to cardiovascular disease and outcome in patients with chronic kidney disease (CKD). Visfatin is an adipocytokine that recently generated much interest; however, its role in CKD remains to be clarified. This study aimed to assess visfatin in correlation with markers of ED and inflammation in Egyptian patients with CKD.
The study included 40 non-diabetic, clinically stable CKD patients and 20 healthy volunteers. Serum levels of visfatin, markers of ED (intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)) and markers of inflammation (interleukin-6 (IL-6), and C-reactive protein (CRP)) were measured. Endothelial function was evaluated using brachial artery flow-mediated dilatation (FMD).
Serum visfatin, ICAM-1, VCAM-1, CRP, and IL-6 levels were significantly elevated and FMD% was decreased in CKD patients as compared to controls. Visfatin correlated positively with ICAM-1, VCAM-1, CRP, and IL-6 and negatively with FMD% in CKD patients. In a multiple regression model, visfatin was strongly and independently associated with FMD (Beta = −0.02, P < 0.001) in CKD patients.
Serum visfatin is strongly associated with endothelial adhesion molecules and FMD%, suggesting that visfatin is an important promising biomarker for prediction of ED and future cardiovascular risk in CKD patients. Moreover, the relationship between visfatin and IL-6 indicates that circulating visfatin may reflect the sub-clinical inflammatory status. Thus, visfatin might be involved in the complex interactions between ED, inflammation, and atherosclerosis and their major clinical consequences; however, further prospective studies are required to prove this hypothesis.
Haptoglobin gene polymorphism in type 2 diabetic patients with and without nephropathy: An Egyptian study
2007, European Journal of Internal Medicine
Citation Excerpt :
One aliquot was ready for measurement of fasting blood glucose, blood urea, and serum creatinine using commercial assay kits supplied by Bicon Co. (Germany) and the creatinine clearance was calculated. The other aliquots were stored at − 20 °C for measurement of serum C-reactive protein (CRP) using a high-sensitivity latex-enhanced immunonephelometric assay (Dade Behring, Germany) [27], serum IL-6 using a commercially available double-sandwich enzyme-linked immunosorbent assay (Quantikine, R&D Systems, Minneapolis, Minn) [28], and serum Hp concentration based on the absorbance of haptoglobin–hemoglobin (Hp–Hb) complex in acid solution pH 3.7 [29]. Haptoglobin phenotype distribution was determined using 5% polyacrylamide gel electrophoresis (PAGE), as previously described [30,31].
The development and progression of diabetic microvascular complications including nephropathy are related to the degree of glycemic control and oxidative stress and may be influenced by genetic factors. The aim of the present study was to investigate the association between haptoglobin (Hp) gene polymorphism and the occurrence of diabetic nephropathy in patients with type 2 diabetes mellitus and to find a possible link between Hp phenotypes and the inflammatory parameters; serum C-reactive protein (CRP), interleukin- 6 (IL-6), and Hp.
The study included 60 normotensive type 2 diabetic patients (> 5 years duration) categorized into three equal groups (normo-, micro-, and macroalbuminuric), according to urinary albumin excretion (UAE). In addition, 20 age- and sex-matched individuals were selected to serve as a control group. Serum CRP, IL-6, and Hp concentrations were measured and Hp phenotyping was conducted using polyacrylamide gel electrophoresis.
The frequency of Hp phenotype 1-1 (Hp 1-1) in diabetic patients with normoalbuminuria was 7/20 (35%) as compared with 1/20 (5%) in diabetics with macroalbuminuria (p = 0.02). However, the frequency of Hp 2-2 was greater in diabetics with macroalbuminuria (12/20, 60%) than in those with normoalbuminuria or controls (5/20, 25%; p = 0.03). Patients with diabetic nephropathy (micro- or macroalbuminuria) had higher levels of serum CRP, IL-6, and Hp than those without nephropathy (normoalbuminuria). Serum Hp levels in type 2 diabetics were higher in Hp phenotype 2-2 than in Hp 1-1; however, serum CRP and IL-6 levels did not differ significantly between Hp phenotype groups. Moreover, there were significant positive correlations between UAE and serum levels of CRP, IL-6, and Hp in diabetic patients.
Hp phenotype 2-2 is considered to be a major susceptibility gene for the development of nephropathy in type 2 diabetic patients. In addition, the significant association between inflammatory parameters and UAE indicates that inflammation may be a pathogenic mechanism of renal injury in type 2 diabetics. Moreover, serum IL-6 and Hp may be good prognostic factors for the development of nephropathy in the course of diabetes mellitus. Future research on the use of anti-inflammatory therapy may result in a new approach to the treatment and prevention of diabetic nephropathy.
Interleukin-6 (IL-6)
2003, The Cytokine Handbook
Decreased soluble interleukin-6 receptor in patients with acute myocardial infarction
1999, American Heart Journal
Background: The plasma level of interleukin-6 (IL-6), a proinflammatory cytokine, has been reported to be elevated in patients with acute myocardial infarction (AMI). In addition to a specific cell-surface IL-6 receptor, a soluble IL-6 receptor (sIL-6R) exists in plasma as an extracellular domain of glycoprotein 80. The pathophysiologic roles of IL-6 and sIL-6R in AMI are still unknown. Methods and Results: We measured plasma levels of IL-6 and sIL-6R in 17 patients with AMI to evaluate changes over time at 11 points in the acute phase and compared them with parameters of inflammation, myocardial injury, and atherosclerosis. IL-6 showed a triphasic increase with peaks at 3 hours (276.2 ± 50.0 pg/mL), 2 days (153.6 ± 35.7 pg/mL), and 5 days (180.7 ± 52.3 pg/mL) after the onset of AMI. sIL-6R had biphasic dips at 12 hours (31.1 ± 4.1 ng/mL) and 3 days (29.9 ± 1.5 ng/mL) after the onset on AMI. The time-dependent changes in IL-6 paralleled those in sIL-6R from onset to 5 days. Thereafter, the changes in IL-6 and sIL-6R varied; that is, IL-6 gradually decreased from 5 days to 4 weeks, whereas sIL-6R gradually increased from 5 days to 4 weeks. Significant positive correlations were observed between the absolute increase in IL-6 and the decrease in sIL-6R and the changes in white blood cell count, erythrocyte sedimentation rate, C-reactive protein, creatine kinase, and lactic dehydrogenase. Neither IL-6 nor sIL-6R strongly correlated with parameters of coronary atherosclerosis. Conclusions: These results demonstrate that IL-6 and sIL-6R are associated with the processes of inflammation and myocardial injury during the acute phase of AMI. (Am Heart J 1999;138:908-15.)
B-CLL cells experimentally infected with EBV enter DNA synthesis, produce cytokines and stimulate T-lymphocytes
1996, Immunology Letters
Several B-chronic lymphocytic leukemia (B-CLL) clones, represented by different patients can be infected with EBV in vitro. A proportion of the cells becomes activated by the virus, but they rarely yield immortalized cell lines. We used cells from two B-CLL patients which differed in sensitivity to EBV infection. After 7 days in culture, we studied the CLL cells exposed to the B-cell activators Staphylococcus aureus, IL-2 and/or to EBV for expression of the activation markers CD23, CD39 and the adhesion and costimulatory molecules CD54 and CD80, for DNA synthesis, for production of various cytokines and for capacity to stimulate autologous and allogeneic T-lymphocytes. Generally the frequency of cells expressing cytokines in the cytoplasm correlated with the activation status of the populations and with their capacity to stimulate T-cells. It is likely that the difference between the clones with regard to sensitivity to the viral infection, is determined by the maturation state of the CLL cells. It may therefore reflect the variation in the response within a normal B-cell population. The results obtained in the present and in our earlier experiments with EBV provide information concerning the events after primary EBV infection in vivo. The T-lymphocyte stimulatory capacity of the infected CLL cells may be considered as an in vitro correlate to the syndrome of infectious mononucleosis. The detection of cytokines in the infected B-CLL cells suggests that their production by the B-blasts contributes to the level of T-lymphocytosis induced by the primary infection.
Polyethylene glycol modification of interleukin-6 enhances its thrombopoietic activity
1995, Journal of Controlled Release
This study was conducted to increase the in vivo thrombopoietic activity of interleukin-6 (IL-6). Recombinant human IL-6 was covalently conjugated with N-succinimidyl succinate monomethoxy polyethylene glycol (PEG). The in vitro bioactivity of the PEG-modified IL-6 was reduced with increase in its degree of PEG modification, but the in vivo thrombopoietic activity of PEG-modified IL-6 was markedly increased compared to unmodified IL-6. In particular, modified IL-6, in which 54% of the 14 lysine amino groups were coupled with PEG, showed > 10 times greater thrombopoietic effect in vivo than unmodified IL-6. The area under the serum concentration curve of PEG-modified IL-6 after subcutaneous injection was > 17 times larger than that of unmodified IL-6. Chemical attachment of PEG to IL-6 thus increased the bioavailability of IL-6, and may facilitate its potential therapeutic use.

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Establishment of strongly neutralizing monoclonal antibody to human interleukin-6 and its epitope analysis

Summary

FEBS Lett.

Clin. Immunol. Immunopathol.

J. Immunol. Methods

J. Exp. Med.

Eur. J. Immunol.

Arth. Rheum.