- Email: [email protected]
Interleukin-23 serum levels in patients affected by peripheral arterial disease
Clinical Biochemistry 45 (2012) 275–278
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Case Report
Interleukin-23 serum levels in patients affected by peripheral arterial disease Antonio David a, Salvatore Saitta b,⁎, Giovanni De Caridi c, Filippo Benedetto c, Mafalda Massara c, Domenica Claudia Risitano a, Francesco Saverio Venuti a, Francesco Spinelli c, Sebastiano Gangemi b, d a
Department of Neurosciences, Psichiatric and Anesthesiological Sciences, University of Messina, Italy School and Unit of Allergy and Clinical Immunology, Department of Human Pathology, University of Messina, Italy Department of Cardiovascular and Thoracic Sciences, University of Messina, Italy d Institute of Biomedicine and Molecular Immunology, National Research Council, Palermo, Italy b c
a r t i c l e
i n f o
Article history: Received 30 September 2011 Received in revised form 22 November 2011 Accepted 9 December 2011 Available online 20 December 2011 Keywords: Cytokines Inflammation Interleukin-23 (IL-23) Lymphocyte T helper-17 (Th-17) Peripheral arterial disease (PAD)
a b s t r a c t Objectives: To clarify whether interleukin (IL)-23 is involved in peripheral arterial disease (PAD). Design and methods: We evaluated IL-23 serum levels, in 29 patients suffering from lower extremity PAD and in 30 healthy subjects. Results: IL-23 serum levels were higher during the three times (T0, T1 and T2) compared to the control group, although only statistically significant for T0 and T2: T0 (15.83± 22.08 vs. 8.08± 8.62 pg ml, p = 0.026), T1 (16.10± 23.71 vs. 8.08± 8.62 pg/ml, p = 0.101), T2 (15.06± 16.72 vs. 8.08± 8.62 pg/ml, p = 0.005). Conclusion: For the first time, our data gives us reason to believe there is an involvement of IL-23 in PAD. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Lower extremity PAD is a common syndrome that affects a large proportion of most worldwide adult populations. Epidemiological studies indicate that about 5% men and 2.5% women 60 years of age or older have symptoms of intermittent claudication [1]. The major cause for lower extremity PAD is atherosclerosis [2]. Moreover elevated levels of C-reactive protein, a serological marker of systemic inflammation, are associated with lower extremity PAD [3]. Interleukin (IL)-23 has an important function in the control of certain intracellular infections as well as a role in the alloreactive TH1 responses in transplant models [4]. Recently it has been demonstrated that IL-23 is mainly involved in the generation of a new subset of effector T cells (Th17) that could be characterized by the ability to produce a unique set of inflammatory cytokines, such as IL-17A, IL-17F, IL-6, and tumor necrosis factor-α (TNF-α) [5]. The latter help to stimulate epithelial cells to produce antimicrobial factors by linking the function of IL-23 in mucosal host defense and the cellular mechanism of IL-23-dependent inflammatory bowel disease. For this reason we are beginning to appreciate ⁎ Corresponding author at: School and Unit of Allergy and Clinical Immunology, Department of Human Pathology, University of Messina, A.O.U. Policlinico “G. Martino”, via Consolare Valeria, 98125 Messina Italy. Fax: + 39 090694773. E-mail addresses: [email protected] (A. David), [email protected] (S. Saitta), [email protected] (G. De Caridi), fi[email protected] (F. Benedetto), [email protected] (M. Massara), [email protected] (D.C. Risitano), [email protected] (F.S. Venuti), [email protected] (F. Spinelli), [email protected] (S. Gangemi).
why IL-23 is so central to the pathogenesis of many autoimmune disorders [6]. The importance of inflammation in atherosclerosis has been well defined for cardiovascular disease. However, there is a difference in the amount of data on the relationship between vascular inflammation and PAD. Thus, the purpose of this study is to clarify whether IL-23 is involved in peripheral arterial disease (PAD). Patients and methods Patients We evaluated IL-23 serum levels and other blood values such as red blood cells, white blood cells, platelets, C-reactive protein (CRP), fibrinogen and creatinine in 29 patients (7 females and 22 males¸ mean age 70 ± 14 years; range 27–88 years) suffering from lower extremity PAD. 28 patients had stage IV, only one had stage III, following the Fontaine's classification. All patients underwent lower extremity bypass surgery (LEB). 22 patients underwent LEB using autogenous vein (saphenous) and 7 using synthetic polytetrafluoroethylene (PTFE) graft material. The serial blood samples were collected in three times in connection with surgical treatment: T0 (24 h before), T1 (24 h after) and T2 (5 days after). We also evaluated IL-23 serum levels in 30 healthy age-sexmatched subjects recruited as controls. Each subject gave a written informed consent to the study.
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2011.12.010
276
Table 1 IL-23 serum levels (pg/ml) in 29 patients suffering from lower extremity PAD. Patient no.
White blood cells (mm3)
Platelets (mm3)
CRP (mg/dl)
Fibrinogen (mg/dl)
Creatinine (mg/dl)
T0
T1
T2
T0
T1
T2
T0
T1
T2
T0
T1
T2
T0
T1
T2
T0
T1
T2
T0
T1
T2
2.25 5.86 10.57 2.35 7.60 12.44 2.50 10.87 2.87 7.08 10.76 1.84 9.62 20.30 2.24 10.02 1.93 5.27 10.27 1.68 23.69 6.25 78.18 87.97 27.95 26.29 1.37 58.89 10.24
3.89 6.97 12.90 5.23 9.67 2.62 6.45 9.64 4.48 9.05 10.93 5.35 10.89 4.00 6.39 16.01 1.56 8.84 12.02 4.17 34.75 9.38 74.33 47.99 6.48 18.66 3.24 110.46 10.64
2.96 10.28 2.25 5.61 10.73 4.44 10.17 13.22 6.80 9.13 3.61 6.43 11.89 9.29 10.39 13.30 5.48 10.77 1.36 4.96 35.32 9.63 72.26 46.63 20.58 15.83 11.87 56.56 15.01
3,990,000 3,850,000 3,180,000 5,310,000 3,240,000 2,590,000 3,400,000 4,550,000 3,490,000 3,880,000 3,910,000 3,990,000 3,320,000 3,940,000 3,550,000 3,910,000 3,650,000 4,180,000 4,190,000 5,100,000 2,690,000 5,370,000 2,760,000 3,620,000 3,910,000 4,280,000 5,450,000 4,700,000 3,410,000
3,330,000 3,820,000 3,040,000 4,260,000 3,270,000 2,360,000 2,560,000 3,880,000 3,300,000 3,420,000 2,960,000 3,110,000 3,170,000 3,750,000 3,510,000 3,480,000 2,760,000 3,770,000 3,770,000 4,280,000 2,920,000 4,480,000 3,570,000 4,100,000 3,980,000 4,270,000 5,090,000 4,470,000 2,890,000
2,950,000 3,130,000 2,760,000 3,590,000 2,840,000 2,700,000 3,240,000 3,110,000 2,720,000 3,350,000 3,150,000 3,110,000 2,670,000 3,380,000 3,460,000 3,770,000 2,500,000 3,720,000 2,980,000 4,030,000 3,010,000 4,560,000 3,220,000 4,450,000 3,760,000 3,640,000 4,950,000 4,120,000 3,580,000
7600 6400 4500 11,900 13,500 8800 5700 7900 9900 8700 11,800 9500 3800 6600 11,400 14,500 11,800 6400 10,900 11,000 16,300 10,100 12,600 6100 25,900 7300 6700 7900 8200
11,600 10,500 4,500 20,000 15,600 10,600 6,700 10,300 11,200 12,500 6,600 11,300 4,300 7,300 14,500 17,800 14,400 11,600 15,700 12,500 14,300 15,100 18,200 7,700 22,300 11,000 9,900 11,400 9,400
7400 6700 3900 9000 13,100 6800 6500 6600 8500 7400 6300 12,300 5300 6500 11,000 12,200 10,700 12,200 15,700 10,200 16,900 13,000 17,800 8400 10,100 9400 5700 14,000 12,400
324,000 592,000 268,000 279,000 275,000 174,000 355,000 201,000 219,000 517,000 266,000 272,000 234,000 173,000 565,000 385,000 339,000 115,000 451,000 356,000 307,000 315,000 184,000 217,000 358,000 158,000 179,000 369,000 488,000
289,000 490,000 197,000 282,000 288,000 165,000 284,000 188,000 207,000 453,000 259,000 258,000 264,000 151,000 462,000 414,000 230,000 144,000 525,000 317,000 246,000 284,000 175,000 258,000 425,000 249,000 146,000 374,000 448,000
276,000 369,000 187,000 322,000 289,000 198,000 307,000 230,000 239,000 534,000 324,000 269,000 230,000 218,000 419,000 379,000 285,000 173,000 235,000 498,000 242,000 272,000 156,000 302,000 433,000 241,000 169,000 273,000 389,000
0 2 1 5 8 3 1 2 1 8 7 17 4 11 6 16 5 1 10 9 7 0 17 13 16 2 0 5 1
14 7 8 7 13 7 8 1 2 9 8 15 3 11 23 25 7 1 9 9 19 0 15 14 26 1 0 9 5
12.2 7.7 3.2 8.9 15.5 5.5 6.3 11.0 7.6 3.7 7.6 18.6 5.3 11.5 25.0 26.0 12.5 1.0 9.1 9.1 8.2 0.1 14.0 9.7 10.0 1.4 2.1 8.0 3.1
354 708 420 360 536 797 333 339 525 654 598 721 275 372 556 519 522 420 709 497 483 423 398 448 765 382 318 284 448
268 568 433 568 546 568 214 262 474 508 474 608 299 517 618 599 304 473 674 473 371 304 406 596 868 330 252 233 267
480 742 448 666 706 771 517 630 539 573 765 697 460 709 695 825 561 450 797 696 441 616 429 689 742 368 489 372 540
0.90 0.70 3.80 1.70 1.70 4.30 0.60 1.00 1.90 0.80 1.20 1.20 6.20 6.10 7.70 5.80 0.80 1.60 1.20 4.50 0.80 0.90 1.80 0.60 1.20 1.30 0.70 1.10 0.80
0.70 0.50 6.80 1.90 1.50 7.20 0.40 1.20 1.80 0.60 0.90 0.80 5.40 5.10 8.80 9.20 1.10 1.90 1.00 4.80 0.60 0.80 1.20 0.50 0.80 0.90 0.60 1.10 0.70
1.00 0.90 6.90 1.60 1.30 7.30 0.50 1.00 2.00 0.50 0.80 1.00 6.10 6.50 8.10 7.90 0.70 1.80 1.00 7.10 0.70 0.80 1.10 0.50 0.70 1.30 0.50 1.60 0.70
IL-23 serum levels and other blood values as red blood cells, white blood cells, platelets, C-reactive protein (CRP), fibrinogen and creatinine in 29 patients suffering from lower extremity PAD.
A. David et al. / Clinical Biochemistry 45 (2012) 275–278
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Red blood cells (mm3)
IL-23 (pg/ml)
A. David et al. / Clinical Biochemistry 45 (2012) 275–278
Laboratory measurements Serum samples were obtained using serum separator tube and allowing samples to dot for 30 min before centrifugation (15 min at approximately 1000 ×g); serum aliquots were stored at −20 °C until assay. IL-23 serum concentrations were measured by a quantitative enzyme immunoassay technique. The assay was performed by using a commercially available kit (R&D System Europe, Abingdon, UK); a microplate reader capable of measuring absorbance at 450 nm (correction wavelength set at 540 nm) was used to measure the intensity of colour developed in each well. Blood count (red blood cells, white blood cells, platelets) was performed on whole blood (ABX Pentra Dx120—HORIBA LTD, Kyoto, Japan). Fibrinogen was performed on serum by photometric reading (CA 7000—Siemens Healthcare Diagnostics Inc., Deerfield Illinois, USA). RCP was perfomed on serum by photometric reading (Modular Analytics Systems D 2400—Roche Diagnostics, Indianapolis, USA). Creatinine was performed on serum blood by photometric reading (Roche/Hitachi Modular Pre-Analytics Plus—Roche Diagnostics, Indianapolis, USA). Statistical analysis The statistical analysis was performed with SPSS for Windows (version 13.0). Data were presented as mean ± standard deviation (SD). Differences between unpaired groups were analyzed by the Kolmogorov– Smirnov test. Differences between two paired groups were analyzed by the sign test. Differences between more than two paired groups were analyzed between Friedman test. Correlation coefficients were calculated with the Spearman rank correlation coefficient test. Relationship between variables was evaluated with the ANOVA linear regression which identified the dependent variable in IL-23 and the independent variables in blood values and in other parameters such as age, sex, allergy, smoking, diabetes, hypertension, ischemic heart disease, autoimmune diseases, chronic renal failure and dialysis. Statistical significance was set at p b 0.05. Results IL-23 serum levels were higher in the three times (T0, T1 and T2) compared to the control group (Tables 1 and 2), although only statistically significant for T0 and T2: T0 (15.83± 22.08 vs. 8.08± 8.62 pg ml, p = 0.026), T1 (16.10 ± 23.71 vs. 8.08 ± 8.62 pg/ml, p = 0.101), T2 (15.06 ± 16.72 vs. 8.08 ± 8.62 pg/ml, p = 0.005). Furthermore there was a statistical difference in IL-23 serum levels in the three times as demonstrated by the Freedman test (p= 0.022), especially T0 vs. T1 p = 0.026, T1 vs. T2 p = 0.063 and T0 vs. T2 p = 0.137 by the sign test (Fig. 1). In patients at three times there were no statistically significant correlations between IL-23 serum levels and the single blood values (red cells, white cells, platelets, CRP, fibrinogen and creatinine), by using ANOVA linear regression, we found a statistically significant relationship between IL-23 serum levels and all blood values at T0 (p = 0.007) and at T2 (p = 0.032), but not at T1. However, we did not find a relationship between IL-23 serum levels and all the other parameters such as age, sex, allergy, smoking, diabetes, hypertension, ischemic heart disease, autoimmune diseases, chronic renal failure and dialysis. Use of drugs and how these could influence the IL-23 serum levels was also considered by using the same linear regression used in the two previous cases that, however, did not show a relationship. The 22 patients undergoing LEB with autogenous vein had IL-23 levels, in the three times, greater than the 7 patients who had synthetic
277
Table 2 IL-23 serum levels (pg/ml) in 30 healthy age-sexmatched subjects recruited as controls. Control no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
8.147 1.001 2.234 4.78 8.596 4.437 6.074 6.283 9.434 1.151 4.934 7.99 8.788 9.184 7.113 6.35 3.641 46.075 6.689 14.255 8.058 5.068 3.294 9.383 11.75 0.876 4.921 25.728 2.934 3.33
IL-23 serum levels (pg/ml) in 30 healthy age-sexmatched subjects recruited as controls.
PTFE graft material, even if not statistically significant (T0 = 18.18 ± 24.62 vs. 8.46± 8.34 pg/ml, p = 0.356; T1= 18.10 ± 26.87 vs. 9.82 ± 5.88 pg/ml, p = 0.968; T2= 16.68 ± 18.90 vs. 9.97 ± 3.79 pg/ml, p = 0.824). IL-23 levels in patients who had synthetic PTFE graft material were comparable to controls (T0 = 8.46± 8.34 vs. 8.08 ± 8.62 pg/ml, p=0.999; T1=9.82±5.88 vs. 8.08±8.62 pg/ml, p=0.145; T2=9.97±3.79 vs. 8.08±8.62 pg/ml, p=0.145), while patients undergoing LEB were higher than controls (T0 = 18.18 ± 24.62 vs. 8.08± 8.62 pg/ml, p = 0.010; T1= 18.10 ± 26.87 vs. 8.08 ± 8.62 pg/ml, p = 0.066; T2= 16.68± 18.90 vs. 8.08 ± 8.62 pg/ml, p = 0.010).
Fig. 1. Bars represent means ± standard deviations of IL-23 serum levels in patients, at the three times (T0, T1 and T2) and in controls. T0 vs. controls (15.83 ± 22.08 vs. 8.08 ± 8.62 pg ml, p = 0.026), T1 vs. controls (16.10 ± 23.71 vs. 8.08 ± 8.62 pg/ml, p = 0.101), T2 vs. controls (15.06 ± 16.72 vs. 8.08 ± 8.62 pg/ml, p = 0.005) by the Kolmogorov–Smirnov test. There was a statistical difference in IL-23 serum levels in the three times as demonstrated by the Freedman test (p = 0.022), especially T0 vs. T1 p = 0.026, T1 vs. T2 p = 0.063 and T0 vs. T1 p = 0.137 by the sign test.
278
A. David et al. / Clinical Biochemistry 45 (2012) 275–278
Discussion
Conclusions
The increase in circulating levels of biochemical markers for vascular inflammation, such as cytokines, chemokines and cell adhesion molecules (CAM), has shown an association with atherosclerosis [7]. Different risk factors for PAD such as smoking, hypercholesterolemia, hypertension, diabetes mellitus, and hyperhomocysteinemia [2] are known to cause endothelial activation, thus promoting the process of atherosclerosis. However, there is not much data on the relationship between vascular inflammation and PAD severity. Nevertheless, in 1997 Baba et al. showed that plasma IL-6 level in legs of patients with PAD exceeded that of those without PAD [8]. In 2000, Testa et al. reported that IL-1β and IL-6 gene expression was markedly up-regulated in hypoperfused skeletal muscle of patients with critical lower limb ischemia [9]. IL-23 is involved in the generation of a new subset of effector T cells (Th17), characterized by the ability to produce a unique set of inflammatory cytokines, such as IL-17A, IL-17F, IL-6 and TNF-α [5]. Furthermore, IL-23 may be placed further upstream in the inflammatory response compared to IL-6, which would be a final effector. Moreover, the protective action of blocking the gene encoding the IL-23 was demonstrated in mice against the ischemia / reperfusion injury [10]. It is clear that IL-23 is a proinflammatory cytokine with the ability to induce Th17 subset that in turn produces a set of inflammatory cytokines including IL-6. Thus as seen previously, it plays an important role in vascular inflammation. These studies, apart from indicating vascular inflammation as a cause of PAD, also suggest that IL-23 might be of importance in both diagnosis and development of specific medical therapies. Our experience has also highlighted how the circulating levels of this cytokine are higher in patients with PAD compared to controls, except at T1. This probably occurs because post-operative stress mechanisms can influence the IL-23 serum levels in close proximity to surgery. Furthermore we found that these serum levels are not affected by drug therapies or other conditions investigated and are not individually correlated with the other parameters researched. We observed higher IL-23 serum levels in patients undergoing LEB with autogenous vein compared to those who had synthetic PTFE graft material, so that the latter values were similar to healthy controls. It must also be considered that the paucity of undergoing PTFE graft material procedure (only 7) may have compromised the statistical outcome. We can hypothesize that, in any case, the difference in retrospectively detected IL-23 levels is probably due to different underlying medical conditions.
For the first time, our findings confirm the involvement of IL-23 in PAD and Th17 cytokine network in PAD. Acknowledgment We would like to thank Ms. A. Donato for editing of the text. References [1] Jelnes R, Gaardsting O, Hougaard Jensen K, Baekgaard N, Tonnesen KH, Schroeder T. Fate in intermittent claudication: outcome and risk factors. Br Med J (Clin Res Ed) 1986;293:1137–40. [2] A.T. Hirsch, Z.J. Haskal, N.R. Hertzer, C.W. Bakal, M.A. Creager, J.L. Halperin, L.F. Hiratzka, W.R. Murphy, J.W. Olin, J.B. Puschett, K.A. Rosenfield, D. Sacks, J.C. Stanley, LM Jr Taylor, C.J. White, J. White, R.A. White, E.M. Antman, S.C. Jr Smith, C.D. Adams, J.L. Anderson, D.P. Faxon, V. Fuster, R.J. Gibbons, S.A. Hunt, A.K. Jacobs, R. Nishimura, J.P. Ornato, R.L. Page, B. Riegel; American Association for Vascular Surgery; Society for Vascular Surgery; Society for Cardiovascular Angiography and Interventions; Society for Vascular Medicine and Biology; Society of Interventional Radiology; ACC/AHA Task Force on Practice Guidelines Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease; American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; Vascular Disease Foundation. ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. Circulation 2006;113:463-654. [3] Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Plasma concentration of C-reactive protein and risk of developing peripheral vascular disease. Circulation 1998;97:425–8. [4] Piccotti JR, Li K, Chan SY, Ferrante J, Magram J, Eichwald EJ, et al. Alloantigenreactive Th1 development in IL-12-deficient mice. J Immunol 1998;160:1132–8. [5] Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 2005;201:233–40. [6] Tato CM, Cua DJ. Reconciling id, ego, and superego within interleukin-23. Immunol Rev 2008;226:103–11. [7] Szmitko PE, Wang CH, Weisel RD. New markers of inflammation and endothelial cell activation: part I. Circulation 2003;108:1917–23. [8] Baba T, Uchimura I, Fujisawa K, Morohoshi M, Asaoka H, Tanaka A, et al. Production of interleukin-6 induced by hypoxia linked to peripheral arterial disease. Ann N Y Acad Sci 1997;15:542–8. [9] Testa M, De Ruvo E, Russo A, Citterio F, Serino F, Mangoni A, et al. Induction of interleukin-1beta and interleukin-6 gene expression in hypoperfused skeletal muscle of patients with peripheral arterial disease. Ital Heart J 2000;1: 64–7. [10] Konoeda F, Shichita T, Yoshida H, Sugiyama Y, Muto G, Hasegawa E, et al. Therapeutic effect of IL-12/23 and their signaling pathway blockade on brain ischemia model. Biochem Biophys Res Commun 2010;402:500–6.
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Case Report
Interleukin-23 serum levels in patients affected by peripheral arterial disease Antonio David a, Salvatore Saitta b,⁎, Giovanni De Caridi c, Filippo Benedetto c, Mafalda Massara c, Domenica Claudia Risitano a, Francesco Saverio Venuti a, Francesco Spinelli c, Sebastiano Gangemi b, d a
Department of Neurosciences, Psichiatric and Anesthesiological Sciences, University of Messina, Italy School and Unit of Allergy and Clinical Immunology, Department of Human Pathology, University of Messina, Italy Department of Cardiovascular and Thoracic Sciences, University of Messina, Italy d Institute of Biomedicine and Molecular Immunology, National Research Council, Palermo, Italy b c
a r t i c l e
i n f o
Article history: Received 30 September 2011 Received in revised form 22 November 2011 Accepted 9 December 2011 Available online 20 December 2011 Keywords: Cytokines Inflammation Interleukin-23 (IL-23) Lymphocyte T helper-17 (Th-17) Peripheral arterial disease (PAD)
a b s t r a c t Objectives: To clarify whether interleukin (IL)-23 is involved in peripheral arterial disease (PAD). Design and methods: We evaluated IL-23 serum levels, in 29 patients suffering from lower extremity PAD and in 30 healthy subjects. Results: IL-23 serum levels were higher during the three times (T0, T1 and T2) compared to the control group, although only statistically significant for T0 and T2: T0 (15.83± 22.08 vs. 8.08± 8.62 pg ml, p = 0.026), T1 (16.10± 23.71 vs. 8.08± 8.62 pg/ml, p = 0.101), T2 (15.06± 16.72 vs. 8.08± 8.62 pg/ml, p = 0.005). Conclusion: For the first time, our data gives us reason to believe there is an involvement of IL-23 in PAD. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Lower extremity PAD is a common syndrome that affects a large proportion of most worldwide adult populations. Epidemiological studies indicate that about 5% men and 2.5% women 60 years of age or older have symptoms of intermittent claudication [1]. The major cause for lower extremity PAD is atherosclerosis [2]. Moreover elevated levels of C-reactive protein, a serological marker of systemic inflammation, are associated with lower extremity PAD [3]. Interleukin (IL)-23 has an important function in the control of certain intracellular infections as well as a role in the alloreactive TH1 responses in transplant models [4]. Recently it has been demonstrated that IL-23 is mainly involved in the generation of a new subset of effector T cells (Th17) that could be characterized by the ability to produce a unique set of inflammatory cytokines, such as IL-17A, IL-17F, IL-6, and tumor necrosis factor-α (TNF-α) [5]. The latter help to stimulate epithelial cells to produce antimicrobial factors by linking the function of IL-23 in mucosal host defense and the cellular mechanism of IL-23-dependent inflammatory bowel disease. For this reason we are beginning to appreciate ⁎ Corresponding author at: School and Unit of Allergy and Clinical Immunology, Department of Human Pathology, University of Messina, A.O.U. Policlinico “G. Martino”, via Consolare Valeria, 98125 Messina Italy. Fax: + 39 090694773. E-mail addresses: [email protected] (A. David), [email protected] (S. Saitta), [email protected] (G. De Caridi), fi[email protected] (F. Benedetto), [email protected] (M. Massara), [email protected] (D.C. Risitano), [email protected] (F.S. Venuti), [email protected] (F. Spinelli), [email protected] (S. Gangemi).
why IL-23 is so central to the pathogenesis of many autoimmune disorders [6]. The importance of inflammation in atherosclerosis has been well defined for cardiovascular disease. However, there is a difference in the amount of data on the relationship between vascular inflammation and PAD. Thus, the purpose of this study is to clarify whether IL-23 is involved in peripheral arterial disease (PAD). Patients and methods Patients We evaluated IL-23 serum levels and other blood values such as red blood cells, white blood cells, platelets, C-reactive protein (CRP), fibrinogen and creatinine in 29 patients (7 females and 22 males¸ mean age 70 ± 14 years; range 27–88 years) suffering from lower extremity PAD. 28 patients had stage IV, only one had stage III, following the Fontaine's classification. All patients underwent lower extremity bypass surgery (LEB). 22 patients underwent LEB using autogenous vein (saphenous) and 7 using synthetic polytetrafluoroethylene (PTFE) graft material. The serial blood samples were collected in three times in connection with surgical treatment: T0 (24 h before), T1 (24 h after) and T2 (5 days after). We also evaluated IL-23 serum levels in 30 healthy age-sexmatched subjects recruited as controls. Each subject gave a written informed consent to the study.
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2011.12.010
276
Table 1 IL-23 serum levels (pg/ml) in 29 patients suffering from lower extremity PAD. Patient no.
White blood cells (mm3)
Platelets (mm3)
CRP (mg/dl)
Fibrinogen (mg/dl)
Creatinine (mg/dl)
T0
T1
T2
T0
T1
T2
T0
T1
T2
T0
T1
T2
T0
T1
T2
T0
T1
T2
T0
T1
T2
2.25 5.86 10.57 2.35 7.60 12.44 2.50 10.87 2.87 7.08 10.76 1.84 9.62 20.30 2.24 10.02 1.93 5.27 10.27 1.68 23.69 6.25 78.18 87.97 27.95 26.29 1.37 58.89 10.24
3.89 6.97 12.90 5.23 9.67 2.62 6.45 9.64 4.48 9.05 10.93 5.35 10.89 4.00 6.39 16.01 1.56 8.84 12.02 4.17 34.75 9.38 74.33 47.99 6.48 18.66 3.24 110.46 10.64
2.96 10.28 2.25 5.61 10.73 4.44 10.17 13.22 6.80 9.13 3.61 6.43 11.89 9.29 10.39 13.30 5.48 10.77 1.36 4.96 35.32 9.63 72.26 46.63 20.58 15.83 11.87 56.56 15.01
3,990,000 3,850,000 3,180,000 5,310,000 3,240,000 2,590,000 3,400,000 4,550,000 3,490,000 3,880,000 3,910,000 3,990,000 3,320,000 3,940,000 3,550,000 3,910,000 3,650,000 4,180,000 4,190,000 5,100,000 2,690,000 5,370,000 2,760,000 3,620,000 3,910,000 4,280,000 5,450,000 4,700,000 3,410,000
3,330,000 3,820,000 3,040,000 4,260,000 3,270,000 2,360,000 2,560,000 3,880,000 3,300,000 3,420,000 2,960,000 3,110,000 3,170,000 3,750,000 3,510,000 3,480,000 2,760,000 3,770,000 3,770,000 4,280,000 2,920,000 4,480,000 3,570,000 4,100,000 3,980,000 4,270,000 5,090,000 4,470,000 2,890,000
2,950,000 3,130,000 2,760,000 3,590,000 2,840,000 2,700,000 3,240,000 3,110,000 2,720,000 3,350,000 3,150,000 3,110,000 2,670,000 3,380,000 3,460,000 3,770,000 2,500,000 3,720,000 2,980,000 4,030,000 3,010,000 4,560,000 3,220,000 4,450,000 3,760,000 3,640,000 4,950,000 4,120,000 3,580,000
7600 6400 4500 11,900 13,500 8800 5700 7900 9900 8700 11,800 9500 3800 6600 11,400 14,500 11,800 6400 10,900 11,000 16,300 10,100 12,600 6100 25,900 7300 6700 7900 8200
11,600 10,500 4,500 20,000 15,600 10,600 6,700 10,300 11,200 12,500 6,600 11,300 4,300 7,300 14,500 17,800 14,400 11,600 15,700 12,500 14,300 15,100 18,200 7,700 22,300 11,000 9,900 11,400 9,400
7400 6700 3900 9000 13,100 6800 6500 6600 8500 7400 6300 12,300 5300 6500 11,000 12,200 10,700 12,200 15,700 10,200 16,900 13,000 17,800 8400 10,100 9400 5700 14,000 12,400
324,000 592,000 268,000 279,000 275,000 174,000 355,000 201,000 219,000 517,000 266,000 272,000 234,000 173,000 565,000 385,000 339,000 115,000 451,000 356,000 307,000 315,000 184,000 217,000 358,000 158,000 179,000 369,000 488,000
289,000 490,000 197,000 282,000 288,000 165,000 284,000 188,000 207,000 453,000 259,000 258,000 264,000 151,000 462,000 414,000 230,000 144,000 525,000 317,000 246,000 284,000 175,000 258,000 425,000 249,000 146,000 374,000 448,000
276,000 369,000 187,000 322,000 289,000 198,000 307,000 230,000 239,000 534,000 324,000 269,000 230,000 218,000 419,000 379,000 285,000 173,000 235,000 498,000 242,000 272,000 156,000 302,000 433,000 241,000 169,000 273,000 389,000
0 2 1 5 8 3 1 2 1 8 7 17 4 11 6 16 5 1 10 9 7 0 17 13 16 2 0 5 1
14 7 8 7 13 7 8 1 2 9 8 15 3 11 23 25 7 1 9 9 19 0 15 14 26 1 0 9 5
12.2 7.7 3.2 8.9 15.5 5.5 6.3 11.0 7.6 3.7 7.6 18.6 5.3 11.5 25.0 26.0 12.5 1.0 9.1 9.1 8.2 0.1 14.0 9.7 10.0 1.4 2.1 8.0 3.1
354 708 420 360 536 797 333 339 525 654 598 721 275 372 556 519 522 420 709 497 483 423 398 448 765 382 318 284 448
268 568 433 568 546 568 214 262 474 508 474 608 299 517 618 599 304 473 674 473 371 304 406 596 868 330 252 233 267
480 742 448 666 706 771 517 630 539 573 765 697 460 709 695 825 561 450 797 696 441 616 429 689 742 368 489 372 540
0.90 0.70 3.80 1.70 1.70 4.30 0.60 1.00 1.90 0.80 1.20 1.20 6.20 6.10 7.70 5.80 0.80 1.60 1.20 4.50 0.80 0.90 1.80 0.60 1.20 1.30 0.70 1.10 0.80
0.70 0.50 6.80 1.90 1.50 7.20 0.40 1.20 1.80 0.60 0.90 0.80 5.40 5.10 8.80 9.20 1.10 1.90 1.00 4.80 0.60 0.80 1.20 0.50 0.80 0.90 0.60 1.10 0.70
1.00 0.90 6.90 1.60 1.30 7.30 0.50 1.00 2.00 0.50 0.80 1.00 6.10 6.50 8.10 7.90 0.70 1.80 1.00 7.10 0.70 0.80 1.10 0.50 0.70 1.30 0.50 1.60 0.70
IL-23 serum levels and other blood values as red blood cells, white blood cells, platelets, C-reactive protein (CRP), fibrinogen and creatinine in 29 patients suffering from lower extremity PAD.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Red blood cells (mm3)
IL-23 (pg/ml)
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Laboratory measurements Serum samples were obtained using serum separator tube and allowing samples to dot for 30 min before centrifugation (15 min at approximately 1000 ×g); serum aliquots were stored at −20 °C until assay. IL-23 serum concentrations were measured by a quantitative enzyme immunoassay technique. The assay was performed by using a commercially available kit (R&D System Europe, Abingdon, UK); a microplate reader capable of measuring absorbance at 450 nm (correction wavelength set at 540 nm) was used to measure the intensity of colour developed in each well. Blood count (red blood cells, white blood cells, platelets) was performed on whole blood (ABX Pentra Dx120—HORIBA LTD, Kyoto, Japan). Fibrinogen was performed on serum by photometric reading (CA 7000—Siemens Healthcare Diagnostics Inc., Deerfield Illinois, USA). RCP was perfomed on serum by photometric reading (Modular Analytics Systems D 2400—Roche Diagnostics, Indianapolis, USA). Creatinine was performed on serum blood by photometric reading (Roche/Hitachi Modular Pre-Analytics Plus—Roche Diagnostics, Indianapolis, USA). Statistical analysis The statistical analysis was performed with SPSS for Windows (version 13.0). Data were presented as mean ± standard deviation (SD). Differences between unpaired groups were analyzed by the Kolmogorov– Smirnov test. Differences between two paired groups were analyzed by the sign test. Differences between more than two paired groups were analyzed between Friedman test. Correlation coefficients were calculated with the Spearman rank correlation coefficient test. Relationship between variables was evaluated with the ANOVA linear regression which identified the dependent variable in IL-23 and the independent variables in blood values and in other parameters such as age, sex, allergy, smoking, diabetes, hypertension, ischemic heart disease, autoimmune diseases, chronic renal failure and dialysis. Statistical significance was set at p b 0.05. Results IL-23 serum levels were higher in the three times (T0, T1 and T2) compared to the control group (Tables 1 and 2), although only statistically significant for T0 and T2: T0 (15.83± 22.08 vs. 8.08± 8.62 pg ml, p = 0.026), T1 (16.10 ± 23.71 vs. 8.08 ± 8.62 pg/ml, p = 0.101), T2 (15.06 ± 16.72 vs. 8.08 ± 8.62 pg/ml, p = 0.005). Furthermore there was a statistical difference in IL-23 serum levels in the three times as demonstrated by the Freedman test (p= 0.022), especially T0 vs. T1 p = 0.026, T1 vs. T2 p = 0.063 and T0 vs. T2 p = 0.137 by the sign test (Fig. 1). In patients at three times there were no statistically significant correlations between IL-23 serum levels and the single blood values (red cells, white cells, platelets, CRP, fibrinogen and creatinine), by using ANOVA linear regression, we found a statistically significant relationship between IL-23 serum levels and all blood values at T0 (p = 0.007) and at T2 (p = 0.032), but not at T1. However, we did not find a relationship between IL-23 serum levels and all the other parameters such as age, sex, allergy, smoking, diabetes, hypertension, ischemic heart disease, autoimmune diseases, chronic renal failure and dialysis. Use of drugs and how these could influence the IL-23 serum levels was also considered by using the same linear regression used in the two previous cases that, however, did not show a relationship. The 22 patients undergoing LEB with autogenous vein had IL-23 levels, in the three times, greater than the 7 patients who had synthetic
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Table 2 IL-23 serum levels (pg/ml) in 30 healthy age-sexmatched subjects recruited as controls. Control no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
8.147 1.001 2.234 4.78 8.596 4.437 6.074 6.283 9.434 1.151 4.934 7.99 8.788 9.184 7.113 6.35 3.641 46.075 6.689 14.255 8.058 5.068 3.294 9.383 11.75 0.876 4.921 25.728 2.934 3.33
IL-23 serum levels (pg/ml) in 30 healthy age-sexmatched subjects recruited as controls.
PTFE graft material, even if not statistically significant (T0 = 18.18 ± 24.62 vs. 8.46± 8.34 pg/ml, p = 0.356; T1= 18.10 ± 26.87 vs. 9.82 ± 5.88 pg/ml, p = 0.968; T2= 16.68 ± 18.90 vs. 9.97 ± 3.79 pg/ml, p = 0.824). IL-23 levels in patients who had synthetic PTFE graft material were comparable to controls (T0 = 8.46± 8.34 vs. 8.08 ± 8.62 pg/ml, p=0.999; T1=9.82±5.88 vs. 8.08±8.62 pg/ml, p=0.145; T2=9.97±3.79 vs. 8.08±8.62 pg/ml, p=0.145), while patients undergoing LEB were higher than controls (T0 = 18.18 ± 24.62 vs. 8.08± 8.62 pg/ml, p = 0.010; T1= 18.10 ± 26.87 vs. 8.08 ± 8.62 pg/ml, p = 0.066; T2= 16.68± 18.90 vs. 8.08 ± 8.62 pg/ml, p = 0.010).
Fig. 1. Bars represent means ± standard deviations of IL-23 serum levels in patients, at the three times (T0, T1 and T2) and in controls. T0 vs. controls (15.83 ± 22.08 vs. 8.08 ± 8.62 pg ml, p = 0.026), T1 vs. controls (16.10 ± 23.71 vs. 8.08 ± 8.62 pg/ml, p = 0.101), T2 vs. controls (15.06 ± 16.72 vs. 8.08 ± 8.62 pg/ml, p = 0.005) by the Kolmogorov–Smirnov test. There was a statistical difference in IL-23 serum levels in the three times as demonstrated by the Freedman test (p = 0.022), especially T0 vs. T1 p = 0.026, T1 vs. T2 p = 0.063 and T0 vs. T1 p = 0.137 by the sign test.
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Discussion
Conclusions
The increase in circulating levels of biochemical markers for vascular inflammation, such as cytokines, chemokines and cell adhesion molecules (CAM), has shown an association with atherosclerosis [7]. Different risk factors for PAD such as smoking, hypercholesterolemia, hypertension, diabetes mellitus, and hyperhomocysteinemia [2] are known to cause endothelial activation, thus promoting the process of atherosclerosis. However, there is not much data on the relationship between vascular inflammation and PAD severity. Nevertheless, in 1997 Baba et al. showed that plasma IL-6 level in legs of patients with PAD exceeded that of those without PAD [8]. In 2000, Testa et al. reported that IL-1β and IL-6 gene expression was markedly up-regulated in hypoperfused skeletal muscle of patients with critical lower limb ischemia [9]. IL-23 is involved in the generation of a new subset of effector T cells (Th17), characterized by the ability to produce a unique set of inflammatory cytokines, such as IL-17A, IL-17F, IL-6 and TNF-α [5]. Furthermore, IL-23 may be placed further upstream in the inflammatory response compared to IL-6, which would be a final effector. Moreover, the protective action of blocking the gene encoding the IL-23 was demonstrated in mice against the ischemia / reperfusion injury [10]. It is clear that IL-23 is a proinflammatory cytokine with the ability to induce Th17 subset that in turn produces a set of inflammatory cytokines including IL-6. Thus as seen previously, it plays an important role in vascular inflammation. These studies, apart from indicating vascular inflammation as a cause of PAD, also suggest that IL-23 might be of importance in both diagnosis and development of specific medical therapies. Our experience has also highlighted how the circulating levels of this cytokine are higher in patients with PAD compared to controls, except at T1. This probably occurs because post-operative stress mechanisms can influence the IL-23 serum levels in close proximity to surgery. Furthermore we found that these serum levels are not affected by drug therapies or other conditions investigated and are not individually correlated with the other parameters researched. We observed higher IL-23 serum levels in patients undergoing LEB with autogenous vein compared to those who had synthetic PTFE graft material, so that the latter values were similar to healthy controls. It must also be considered that the paucity of undergoing PTFE graft material procedure (only 7) may have compromised the statistical outcome. We can hypothesize that, in any case, the difference in retrospectively detected IL-23 levels is probably due to different underlying medical conditions.
For the first time, our findings confirm the involvement of IL-23 in PAD and Th17 cytokine network in PAD. Acknowledgment We would like to thank Ms. A. Donato for editing of the text. References [1] Jelnes R, Gaardsting O, Hougaard Jensen K, Baekgaard N, Tonnesen KH, Schroeder T. Fate in intermittent claudication: outcome and risk factors. Br Med J (Clin Res Ed) 1986;293:1137–40. [2] A.T. Hirsch, Z.J. Haskal, N.R. Hertzer, C.W. Bakal, M.A. Creager, J.L. Halperin, L.F. Hiratzka, W.R. Murphy, J.W. Olin, J.B. Puschett, K.A. Rosenfield, D. Sacks, J.C. Stanley, LM Jr Taylor, C.J. White, J. White, R.A. White, E.M. Antman, S.C. Jr Smith, C.D. Adams, J.L. Anderson, D.P. Faxon, V. Fuster, R.J. Gibbons, S.A. Hunt, A.K. Jacobs, R. Nishimura, J.P. Ornato, R.L. Page, B. Riegel; American Association for Vascular Surgery; Society for Vascular Surgery; Society for Cardiovascular Angiography and Interventions; Society for Vascular Medicine and Biology; Society of Interventional Radiology; ACC/AHA Task Force on Practice Guidelines Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease; American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; Vascular Disease Foundation. ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. Circulation 2006;113:463-654. [3] Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Plasma concentration of C-reactive protein and risk of developing peripheral vascular disease. Circulation 1998;97:425–8. [4] Piccotti JR, Li K, Chan SY, Ferrante J, Magram J, Eichwald EJ, et al. Alloantigenreactive Th1 development in IL-12-deficient mice. J Immunol 1998;160:1132–8. [5] Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 2005;201:233–40. [6] Tato CM, Cua DJ. Reconciling id, ego, and superego within interleukin-23. Immunol Rev 2008;226:103–11. [7] Szmitko PE, Wang CH, Weisel RD. New markers of inflammation and endothelial cell activation: part I. Circulation 2003;108:1917–23. [8] Baba T, Uchimura I, Fujisawa K, Morohoshi M, Asaoka H, Tanaka A, et al. Production of interleukin-6 induced by hypoxia linked to peripheral arterial disease. Ann N Y Acad Sci 1997;15:542–8. [9] Testa M, De Ruvo E, Russo A, Citterio F, Serino F, Mangoni A, et al. Induction of interleukin-1beta and interleukin-6 gene expression in hypoperfused skeletal muscle of patients with peripheral arterial disease. Ital Heart J 2000;1: 64–7. [10] Konoeda F, Shichita T, Yoshida H, Sugiyama Y, Muto G, Hasegawa E, et al. Therapeutic effect of IL-12/23 and their signaling pathway blockade on brain ischemia model. Biochem Biophys Res Commun 2010;402:500–6.