| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Medicine II (A.K., T.H., H.Mat., Y.M., M.O., N.T., H.Mas., M.I.-S., M.N., T.I.), Kansai Medical University, Moriguchi, Osaka 570-8506, Japan; Advanced Life Science Institute Inc. (T.K., K.I.), Wako, Saitama 351-0112, Japan; and Department of Anatomy I (J.H.), Iwate Medical University School of Medicine, Morioka, Iwate 020-8505, Japan
Address all correspondence and requests for reprints to: Atsushi Kosaki, M.D., Ph.D., the Department of Medicine II, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8506, Japan. E-mail: kosakia{at}takii.kmu.ac.jp.
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
B in vascular endothelial cells, smooth muscle cells, and monocytes (2, 3, 4, 5). AGE also activates the Janus kinase/signal transducer and activator of transcription kinase pathway via RAGE and increases collagen production in kidney cells (6). These results indicate that the RAGE pathway plays an important role in the development of diabetic complications and accelerated atherosclerosis. Extracellular newly identified RAGE-binding protein (EN-RAGE) purified from bovine lung extract has been reported as a ligand for RAGE (7). Analysis of its sequence indicated that EN-RAGE was the bovine counterpart of human S100A12, also known as calcium-binding protein in amniotic fluid-1 (CAAF1) or calgranulin C, a member of the S100 protein family, which is comprised of a closely related group of low-molecular-weight (10–14 kDa) acidic calcium-binding proteins with elongation factor-hands (8). S100A12 protein is abundantly expressed in the esophageal epithelium, neutrophils, and monocytes/macrophages in human (9, 10). The human S100A12 gene is mapped to chromosome 1q21.2-q22, in which most of the S100 genes form a cluster, and consists of three exons with the two elongation factor-hand motifs encoded separately by exons 2 and 3 (11). The sequence of the 92-amino acid human S100A12 shares 40, 46, 66, and 70% identity with human S100A8 (calgranulin A), human S100A9 (calgranulin B), bovine S100A12 (EN-RAGE), and pig S100A12 (calgranulin C), respectively.
Engagement of RAGE by S100A12 also activates nuclear factor-B, a central transcription factor for inflammatory events, and induces adhesion molecules such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in vascular endothelial cells (7). It also mediates the migration and activation of monocytes (12), indicating their potential contribution to the development of micro- and macroangiopathy. Indeed, a recent report has shown that serum concentrations of S100A12 were elevated in the patients with overt systemic vasculitis (Kawasaki disease) and associated with the active signs of acute vasculitis (13). Therefore, S100A12 protein seems to be a new marker for inflammation.
Recently, subclinical inflammation has attracted much attention as a contributor to diabetic micro- and macroangiopathy. In this regard, experimental evidence and cross-sectional data suggest that IL-6 and C-reactive protein (CRP), both markers for inflammation, correlate with hyperglycemia, insulin resistance, and development of type 2 diabetes (14). Diabetes also elevates plasma levels of TNF
and plasminogen activator inhibitor (PAI)-1 (15, 16, 17). However, little is known regarding the modulation of the proinflammatory and atherogenic cytokine-like protein S100A12 in patients with diabetes mellitus.
In this study, we developed a high-sensitivity ELISA system using specific monoclonal antibodies against recombinant human S100A12 to determine plasma S100A12 levels in patients with diabetes mellitus. With the use of this ELISA system, we have demonstrated for the first time that patients with type 2 diabetes have increased plasma S100A12 protein levels. Moreover, statistic analyses revealed that hemoglobin A1c (HbA1c) and white blood cell count (WBC) are significant independent determinants of plasma S100A12 concentration.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Subjects
The following groups were studied: 40 patients with type 2 diabetes (19 men and 21 women) and 35 patients without diabetes (21 men and 14 women) in Kansai Medical University Hospital. Among the diabetic patients, five were treated with diet and exercise, 23 with oral hypoglycemic agents, and 12 with insulin. All subjects had no clinical evidence of overt infection, chronic inflammatory diseases, or diabetic ketoacidosis. Patients with renal dysfunction (serum creatinine >1.0 mg/dl) were excluded. Blood samples for the assay were withdrawn from the antecubital vein and immediately transferred to tubes containing Na2EDTA (1 mg/ml) and centrifuged at 4 C. The plasma was immediately frozen and stored at –80 C until the time of assay.
Laboratory methods
Low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) levels were measured using enzymatic colorimetric methods. HbA1c was measured using a latex-enhanced immunoassay. Blood glucose was measured using a glucose-oxidation method. High-sensitivity (hs) C-reactive protein was measured by N Latex high sensitivity CRP kit (Dade Behring Inc., Deerfield, IL).
Expression and purification of recombinant human S100A12
Human S100A12 cDNA was cloned as described previously (11). Recombinant S100A12 was expressed in Escherichia coli as a glutathione-S-transferase fusion protein. The fusion protein was affinity purified using glutathione-Sepharose 4B according to the manufacturer’s protocol (Amersham Biosciences Corp., Piscataway, NJ). The fusion protein was digested by factor Xa. The rS100A12 was separated from glutathione-S-transferase by gel filtration on a Superdex 200-pg column (Amersham).
Production of monoclonal antibodies
Female BALB/c mice were immunized ip with the purified recombinant human S100A12 protein in Freund’s adjuvant (Wako Pure Chemical Industries, Osaka, Japan). The mouse splenocytes were fused with SP2/0-Ag14 myeloma cells. Anti-S100A12 monoclonal antibody producing hybridomas were selected by rhS100A12-coated ELISA and cloned by limiting dilution. The specificity of the monoclonal antibodies (mAbs) was also determined by an immunoblot method. The immunoglobulin subclasses of mAbs were determined with a mouse MonoAb-ID kit (Zymed, San Francisco, CA). All hybridoma cell lines were transplanted into the mouse abdominal cavity. The ascitic fluids were collected, and the mAbs were purified by protein-A column chromatography (Amersham).
ELISA for S100A12 protein quantification
For the quantification of S100A12 protein in human plasma, a sandwich assay was performed with mAb hCF128 and horseradish peroxidase (HRPO)-labeled mAb hCF113. The hCF128 reacted with S100A12 independent of Ca2+, whereas hCF113 recognized the Ca2+ binding forms of S100A12. Monoclonal antibody hCF113 were digested by pepsin (Worthington Biochemical Corp., Freehold, NJ) in 100 mM acetate buffer (pH 4.0), and the F(ab')2 fragment was isolated by gel filtration on Superdex 200HR (Amersham). The Fab' fragment was prepared by reducing the F(ab')2 fragment and was conjugated to HRPO (Toyobo, Osaka, Japan) by the maleimide hinge method (18). The conjugate was purified by gel filtration chromatography on Superdex 200HR. Microtiter wells (MaxiSorp F8; Nunc, Roskilde, Denmark) were coated with 100 µl mAb hCF128 (0.3 µg/ml). Thereafter, the wells were washed twice with HEPES-buffered saline [HBS; 0.15 M NaCl, 20 mM HEPES (pH 7.4)] followed by incubation with blocking solution [1% sucrose, 1% BSA in HBS (pH 7.4)] at room temperature for 2 h. After removing the blocking solution, the wells were vacuum dried and stored at 4 C until the assay.
A 10-µl specimen aliquot was added to each well filled with 100 µl assay buffer [1% BSA, 0.05% Tween 20, 1 mM EDTA in HBS (pH 7.5)]. The wells were incubated for 1 h at room temperature and then washed three times with washing buffer [0.05% Tween 20 in HBS (pH 7.4)]. A 100-µl aliquot of HRPO-conjugated hCF113 antibody solution (0.1 µg/ml) in conjugate buffer [1% BSA, 0.05% Tween 20, 1 mM CaCl2 in HBS (pH 7.5)] was added to each well and incubated for 1 h at room temperature. After washing four times with washing buffer, 100 µl of substrate solution [0.4 µg/ml of o-phenylenediamine and 0.006% H2O2 in 100 mM citrate buffer (pH 5.6)] were added and incubated for 30 min at room temperature. After the enzyme reaction was stopped with 100 µl of 1 M H2SO4, the absorbance at 492 nm was measured by a microplate reader. The concentration of S100A12 in the samples was obtained by interpolation of their absorbance from the calibration curve.
Western blotting
One milliliter of plasma sample was preabsorbed with protein A-Sepharose F.F. (Amersham). The supernatant was immunoprecipitated with 5 µg anti-S100A12 mAbs (hCF128) at room temperature for 1 h, followed by the addition of protein A-Sepharose F.F. at room temperature for 1 h. The Sepharose-bound immune complexes were collected by centrifugation (10,000 x g for 1 min) and then washed three times in washing buffer. After the immunoprecipitates were denatured in Laemmli’s sample buffer by boiling for 5 min, proteins were separated by electrophoresis on 15–25% Tricine/SDS-PAGE and transferred to polyvinyl difluoride membranes (Immobilon P; Millipore, Bedford, MA) by the semidry blot method. The membranes were blocked by Block Ace (Snow Brand, Sapporo, Japan) solution, subsequently incubated with 1 µg/ml of anti-S100A12 mAbs (biotin-labeled hCF113) at room temperature for 1 h. After several washes in HBS containing 0.05% Tween 20, the membranes were incubated with alkaline phosphatase-conjugated streptavidin (Roche Molecular Biochemical, Mannheim, Germany) at room temperature for 1 h. Antibody binding was visualized using the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium chloride solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
Statistical analysis
Results are expressed as mean ± SE except Fig. 1
. Statistical analyses of the data were performed using an unpaired t test, Mann-Whitney U test, Fisher’s exact probability test, and univariate and stepwise multiple regression analyses, as appropriate, with the use of StatView software (version 5.01; SAS Institute, Cary, NC). A value of P < 0.05 was considered significant.
|
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
As the result of fusion cell screening, 21 clones of monoclonal antibodies against human S100A12 were established. All mAbs are of subclass IgG1. Among them, mAb hCF113 and mAb hCF128 were selected to develop the sandwich ELISA because preliminary experiments had shown that they provided the greatest sensitivity. A representative standard curve and specificity based on recombinant S100A12 calibrators is shown in Fig. 1
. The ELISA system we developed shows no cross-reaction with S100A8, S100A9, and S100B, which have the greatest amino acid sequence homology against S100A12 (Fig. 1A). The absorbance at 492 nm against the amount of calibrator exhibited a linear relationship within the range of 0.1–30 ng/ml (Fig. 1, B and C). The analytical lower detection limit was 0.2 ng/ml, at which the mean –2 SD did not overlap with the mean +2 SD of the zero calibrator in 10 assays. The dilution curves of plasma samples were parallel to that observed using recombinant S100A12 calibrator, indicating that the structure of the active materials present in these samples was immunologically indistinguishable from that of recombinant S100A12 (Fig. 1C). Moreover, intraassay reproducibility was assessed from 10 measurements of five specimens, and the coefficients of variation were less than 4% (1.24, 3.76, 1.92, 1.93, and 3.41%). Interassay reproducibility was assessed from four assays of five specimens, and the coefficients of variation of all were less than 9% (8.51, 3.62, 2.76, 4.60, and 2.83%). In the preliminary test with the use of this ELISA system, we measured concentrations of S100A12 in the plasma of 42 healthy controls who underwent routine blood tests. Among these individuals, we noted no differences in concentration of S100A12 between people of different age and gender. The mean concentration of plasma S100A12 was 10.7 ± 0.96 ng/ml. We also have reported that S100A12 protein in cultured medium is detected by this ELISA system (19).
Finally, to exclude any possible unspecific cross-reactivity of the two antibodies with components of healthy and diabetic plasma, Western blotting of plasma samples was performed with the anti-S100A12 antibodies used for the ELISA system (Fig. 1D). Predictive 7-kDa band for S100A12 protein, however, was not detected directly in the plasma by Western blotting (Fig. 1D, lane 2) probably because of the sensitivity (the sensitivity of our Western blots were > 100 ng recombinant S100A12/lane). In the preliminary experiments, it was found that concentration of S100A12 protein is increased in plasma sample leaving at room temperature. Therefore, S100A12 rich plasma was prepared by leaving at room temperature and used for Western blotting (Fig. 1D, lanes 3–6). As expected, the 7-kDa band was detected when the concentration of S100A12 was more than approximately 100 ng/ml. Densities of bands by Western blotting were comparable with the concentrations of plasma S100A12 measured by the ELISA system. Moreover, no unspecific cross-reactivity with components of normal and diabetic plasma was observed.
Plasma S100A12 concentrations in patients with type 2 diabetes
The clinical parameters of the patients are shown in Table 1 as mean ± SE. HbA1c, fasting glucose, TG, and hsCRP levels were significantly higher in the patients with diabetes (DM group), compared with those without diabetes (non-DM group). No differences were observed in gender, age, incidence of smoker, body mass index (BMI), LDL-C, HDL-C, and WBC. The mean of plasma S100A12 levels was twice as high in the DM group, compared with the non-DM group (19.6 ± 5.26 vs. 8.1 ± 0.81 ng/ml, P < 0.001) (Fig. 2). Moreover, plasma S100A12 concentrations correlated with HbA1c (P < 0.001, R = .455), fasting glucose (P = 0.019, R = .281), hsCRP (P = 0.048, R = .223), and WBC (P = 0.007, R = .325) on univariate analysis in all subjects (Figs. 3 and 4) but not with age, BMI, TG, LDL-C, and HDL-C. Stepwise multiple regression analyses, however, revealed that only HbA1c (P = 0.0001, R = .467) and WBC (P = 0.0001, R = .316) remained significant independent determinants of plasma S100A12 concentration (Table 2).
|
|
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The past decade has been characterized by a growing interest in the idea that atherosclerosis is a subclinical chronic inflammatory disease. Subclinical chronic systemic inflammatory processes have been shown to be characterized by increased circulating levels of proinflammatory cytokines (IL-6, TNF, IL-1ß) and cytokine-responsive acute phase proteins such as CRP, PAI-1, and fibrinogen. Many studies have shown that elevated concentrations of these acute-phase reactants are found in patients with atherosclerosis-related diseases, i.e. acute coronary syndromes and diabetes mellitus (14, 15, 16, 17, 27). Moreover, we have recently shown that the production of S100A12 is induced by IL-6 and inhibited by the activation of peroxisomal proliferator-activated receptor-
by a thiazolidinedione in human macrophages (19). Kislinger et al. (28) also reported that diabetic aortas and kidney displayed increased expression of S100A12, compared with nondiabetic subjects, suggesting that S100A12 may contribute to diabetic microangiopathy and accelerated atherosclerosis. However, little is known regarding the modulation of S100A12 levels in patients with diabetes.
In the present study, we have reported the development of a hsELISA system using specific monoclonal antibodies against recombinant human S100A12 for use in determining plasma S100A12 levels in patients. With this ELISA system, we have demonstrated that plasma S100A12 levels are twice as high in the patients with diabetes, compared with those without diabetes. Moreover, stepwise multiple regression analyses revealed that both HbA1c and WBC remained significant independent determinants of plasma S100A12 concentration, suggesting that granulocytes and monocytes are the sources of plasma S100A12 even in subjects without overt inflammation.
In the meantime, plasma S100A12 protein level is supposed to be regulated by another factor related to blood glucose level. Statistic analyses showed that plasma S100A12 levels correlate with HbA1c more than fasting glucose levels, suggesting that turnover time of S100A12 protein in plasma is relatively slow. The mechanism by which the glucose-related factor causes increased plasma S100A12 protein levels is unknown. Hyperglycemia, however, might stimulate S100A12 protein production directly in granulocytes, monocytes, or other cells. Another possibility is that S100A12 protein production may be up-regulated via increased cytokines and/or other molecules production by hyperglycemia. In this regard, experimental evidence and cross-sectional data have been shown that PAI-1, TNF, and IL-6 are elevated in patients with diabetes (14, 15, 16, 17). Among these molecules, TNF and IL-6 are shown to up-regulate S100A12 expression in monocytes and macrophages (12, 19). Therefore, they may be the link between hyperglycemia and increased plasma S100A12 in patients with diabetes. Further study will be necessary to clarify this point.
Finally, we evaluated the relationship between plasma S100A12 level and existence of diabetic complications. So far, no difference in plasma S100A12 levels has been found in patients with and without diabetic retinopathy, nephropathy, or neuropathy (data not shown). This may be because of the scatter of the duration of diabetes at the time of assessments in this cross-sectional study. Therefore, we should continue to monitor the patients to clarify the relationship between plasma S100A12 level and developments of diabetic complications and accelerated atherosclerosis.
In summary, we demonstrated that plasma S100A12 levels were more than twice as high in patients with diabetes, compared with those without diabetes. Stepwise multiple regression analyses revealed that both HbA1c and WBC are significant independent determinants of plasma S100A12 concentration. These results suggest that the plasma S100A12 protein level was regulated by factors related to subclinical inflammation and glucose control in patients with type 2 diabetes.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
A.K., T.H., and T.K. contributed equally to the work.
Abbreviations: AGE, Advanced glycation end products; BMI, body mass index; CRP, C-reactive protein; EN-RAGE, extracellular newly identified RAGE-binding protein; HbA1c, hemoglobin A1c; HBS, HEPES-buffered saline; HDL-C, high-density lipoprotein cholesterol; HRPO, horseradish peroxidase; hs, high sensitivity; LDL-C, low-density lipoprotein cholesterol; mAb, monoclonal antibody; PAI, plasminogen activator inhibitor; RAGE, receptor for advanced glycation end products; TG, triglyceride; WBC, white blood cell count.
Received December 31, 2003.
Accepted August 4, 2004.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
)-(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression. J Biol Chem 274:31740–31749B transcriptional activation and cytokine secretion. Diabetes 50:1495–1504 are increased in obese patients with noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 83:859–862 concentrations in a native Canadian population with high rates of type 2 diabetes mellitus. J Clin Endocrinol Metab 84:272–278This article has been cited by other articles:
 |
|
 |
|
  M. Kalousova, M. Jachymova, O. Mestek, M. Hodkova, M. Kazderova, V. Tesar, and T. Zima Receptor for advanced glycation end products--soluble form and gene polymorphisms in chronic haemodialysis patients Nephrol. Dial. Transplant., July 1, 2007; 22(7): 2020 - 2026. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Ding, A. Kantarci, J. A. Badwey, H. Hasturk, A. Malabanan, and T. E. Van Dyke Phosphorylation of Pleckstrin Increases Proinflammatory Cytokine Secretion by Mononuclear Phagocytes in Diabetes Mellitus J. Immunol., July 1, 2007; 179(1): 647 - 654. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Ding, A. Kantarci, H. Hasturk, P. C. Trackman, A. Malabanan, and T. E. Van Dyke Activation of RAGE induces elevated O2- generation by mononuclear phagocytes in diabetes J. Leukoc. Biol., February 1, 2007; 81(2): 520 - 527. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Foell, H. Wittkowski, T. Vogl, and J. Roth S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules J. Leukoc. Biol., January 1, 2007; 81(1): 28 - 37. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Basta, A. M. Sironi, G. Lazzerini, S. Del Turco, E. Buzzigoli, A. Casolaro, A. Natali, E. Ferrannini, and A. Gastaldelli Circulating Soluble Receptor for Advanced Glycation End Products Is Inversely Associated with Glycemic Control and S100A12 Protein J. Clin. Endocrinol. Metab., November 1, 2006; 91(11): 4628 - 4634. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Koyama, T. Shoji, H. Yokoyama, K. Motoyama, K. Mori, S. Fukumoto, M. Emoto, T. Shoji, H. Tamei, H. Matsuki, et al. Plasma Level of Endogenous Secretory RAGE Is Associated With Components of the Metabolic Syndrome and Atherosclerosis Arterioscler. Thromb. Vasc. Biol., December 1, 2005; 25(12): 2587 - 2593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. A. Buhimschi, C. S. Buhimschi, C. P. Weiner, T. Kimura, B. D. Hamar, A. K. Sfakianaki, E. R. Norwitz, E. F. Funai, and E. Ratner Proteomic but Not Enzyme-Linked Immunosorbent Assay Technology Detects Amniotic Fluid Monomeric Calgranulins from Their Complexed Calprotectin Form Clin. Vaccine Immunol., July 1, 2005; 12(7): 837 - 844. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
