Skip to main content
SpringerLink
Log in

Anti-tumor Necrosis Factor Alpha (Infliximab) Attenuates Apoptosis, Oxidative Stress, and Calcium Ion Entry Through Modulation of Cation Channels in Neutrophils of Patients with Ankylosing Spondylitis

  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Abstract

Ankylosing Spondylitis (AS) is known to be associated with increased neutrophil activation and oxidative stress, however, the mechanism of neutrophil activation is still unclear. We have hypothesized that the antioxidant and anti-tumor necrosis factor properties of infliximab may affect intracellular Ca2+ concentration in the neutrophils of AS patients. The objective of this study was to investigate the effects of infliximab on calcium signaling, oxidative stress, and apoptosis in neutrophils of AS patients. Neutrophils collected from ten patients with AS and ten healthy controls were used in the study. In a cell viability test, the ideal non-toxic dose and incubation time of infliximab were found as 100 μM and 1 h, respectively. In some experiments, the neutrophils were incubated with the voltage-gated calcium channel (VGCC) blockers verapamil + diltiazem (V + D) and the TRPM2 channel blocker 2-aminoethyl diphenylborinate (2-APB). Intracellular Ca2+ concentration, lipid peroxidation, apoptosis, caspase 3, and caspase 9 values were high in neutrophils of AS patients and were reduced with infliximab treatment. Reduced glutathione level and glutathione peroxidase activity were low in the patients and increased with infliximab treatment. The intracellular Ca2+ concentrations were low in 2-APB and V + D groups. In conclusion, the current study suggests that infliximab is useful against apoptotic cell death and oxidative stress in neutrophils of patients with AS, which seem to be dependent on increased levels of intracellular Ca2+ through activation of TRPM2 and VGCC.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

[Ca2+]i :

Intracellular Ca2+

2-APB:

2-aminoethyl diphenylborinate

AS:

Ankylosing spondylitis

CRP:

C-reactive protein

DMSO:

Dimethyl sulfoxide

EGTA:

Ethylene glycol-bis[2-aminoethylether]-N,N,N,N-tetraacetic acid

ESR:

Erythrocyte sedimentation rate

GSH:

Reduced glutathione

GSH-Px:

Glutathione peroxidase

HBSS:

Hank’s buffered salt solution

ROS:

Reactive oxygen species

SDU:

Suleyman Demirel University

TNF-α:

Tumor necrosis factor alpha

TRP:

Transient receptor potential

TRPM2:

Transient receptor potential melastatin 2

V + D:

Verapamil + diltiazem

References

  • Altintas N, Erboga M, Aktas C, Bilir B, Aydin M, Sengul A, Ates Z, Topcu B, Gurel A (2016) Protective effect of infliximab, a tumor necrosis factor-alfa inhibitor, on bleomycin-induced lung fibrosis in rats. Inflammation 39:65–78

    Article  CAS  PubMed  Google Scholar 

  • Ayub K, Hallett MB (2004) Ca2+ influx shutdown during neutrophil apoptosis: importance and possible mechanism. Immunology 1:8–12

    Article  Google Scholar 

  • Boyraz I, Koç B, Boyacı A, Tutoğlu A, Sarman H, Ozkan H (2014) Ratio of neutrophil/lymphocyte and platelet/lymphocyte in patient with ankylosing spondylitis that are treating with anti-TNF. Int J Clin Exp Med 2014(7):2912–2915

    Google Scholar 

  • Bréchard S, Tschirhart EJ (2008) Regulation of superoxide production in neutrophils: role of calcium influx. J Leukoc Biol 84:1223–1237

    Article  PubMed  PubMed Central  Google Scholar 

  • Couto D, Ribeiro D, Freitas M, Gomes A, Lima JL, Fernandes E (2010) Scavenging of reactive oxygen and nitrogen species by the prodrug sulfasalazine and its metabolites 5-aminosalicylic acid and sulfapyridine. Redox Rep 15:259–267

    Article  CAS  PubMed  Google Scholar 

  • Dalaklioglu S, Tasatargil A, Kale S, Tanriover G, Dilmac S, Erin N (2013) Metastatic breast carcinoma induces vascular endothelial dysfunction in Balb-c mice: role of the tumor necrosis factor-α and NADPH oxidase. Vasc Pharmacol 59:103–111

    Article  CAS  Google Scholar 

  • den Broeder AA, Wanten GJ, Oyen WJ, Naber T, van Riel PL, Barrera P (2003) Neutrophil migration and production of reactive oxygen species during treatment with a fully human anti-tumor necrosis factor-alpha monoclonal antibody in patients with rheumatoid arthritis. J Rheumatol 2003(30):232–237

    Google Scholar 

  • Espino J, Bejarano I, Paredes SD, Barriga C, Reiter RJ, Pariente JA, Rodríguez AB (2011) Melatonin is able to delay endoplasmic reticulum stress-induced apoptosis in leukocytes from elderly humans. Age (Dordr). 33(4):497–507

    Article  CAS  PubMed  Google Scholar 

  • Espino J, Rodríguez AB, Pariente JA (2013) The inhibition of TNF-α-induced leucocyte apoptosis by melatonin involves membrane receptor MT1/MT2 interaction. J Pineal Res 54:442–452

    CAS  PubMed  Google Scholar 

  • Feijóo M, Túnez I, Tasset I, Montilla P, Ruiz A, Collantes E (2009) Infliximab reduces oxidative stress in ankylosing spondylitis. Clin Exp Rheumatol 27:167–168

    PubMed  Google Scholar 

  • Genovese T, Mazzon E, Crisafulli C, Di Paola R, Muià C, Esposito E, Bramanti P, Cuzzocrea S (2008) TNF-alpha blockage in a mouse model of SCI: evidence for improved outcome. Shock 29:32–41

    CAS  PubMed  Google Scholar 

  • Gerlach UA, Atanasov G, Wallenta L, Polenz D, Reutzel-Selke A, Kloepfel M, Jurisch A, Marksteiner M, Loddenkemper C, Neuhaus P, Sawitzki B, Pascher A (2014) Short-term TNF-alpha inhibition reduces short-term and long-term inflammatory changes post-ischemia/reperfusion in rat intestinal transplantation. Transplantation 97:732–739

    Article  CAS  PubMed  Google Scholar 

  • González-Flores D, Rodríguez AB, Pariente JA (2014) TNFα-induced apoptosis in human myeloid cell lines HL-60 and K562 is dependent of intracellular ROS generation. Mol Cell Biochem 390:281–287

    Article  PubMed  Google Scholar 

  • Grynkiewicz C, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450

    CAS  PubMed  Google Scholar 

  • Heiner I, Eisfeld J, Warnstedt M, Radukina N, Jüngling E, Lückhoff A (2006) Endogenous ADP-ribose enables calcium-regulated cation currents through TRPM2 channels in neutrophil granulocytes. Biochem J 398:225–232

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kageyama Y, Takahashi M, Ichikawa T, Torikai E, Nagano A (2008) Reduction of oxidative stress marker levels by anti-TNF-alpha antibody, infliximab, in patients with rheumatoid arthritis. Clin Exp Rheumatol 26:73–80

    CAS  PubMed  Google Scholar 

  • Kaplan Ö, Nazıroğlu M, Güney M, Aykur M (2013) Non-steroidal anti-inflammatory drug modulates oxidative stress and calcium ion levels in the neutrophils of patients with primary dysmenorrhea. J Reprod Immunol 100:87–92

    Article  CAS  PubMed  Google Scholar 

  • Karkucak M, Capkin E, Alver A, Akyuz A, Kiris A, Ak E, Topbas M, Tosun M (2010) The effect of anti-TNF agent on oxidation status in patients with ankylosing spondylitis. Clin Rheumatol 29:303–307

    Article  PubMed  Google Scholar 

  • Kettritz R, Scheumann J, Xu Y, Luft FC, Haller H (2002) TNF-alpha-accelerated apoptosis abrogates ANCA-mediated neutrophil respiratory burst by a caspase-dependent mechanism. Kidney Int 61:502–515

    Article  CAS  PubMed  Google Scholar 

  • Korkmaz S, Erturan I, Nazıroğlu M, Uğuz AC, Ciğ B, Övey IS (2011) Colchicine modulates oxidative stress in serum and neutrophil of patients with Behçet disease through regulation of Ca2+ release and antioxidant system. J Membr Biol 244:113–120

    Article  CAS  PubMed  Google Scholar 

  • Köse SA, Nazıroğlu M (2015) N-acetyl cysteine reduces oxidative toxicity, apoptosis, and calcium entry through TRPV1 channels in the neutrophils of patients with polycystic ovary syndrome. Free Radic Res 49:338–346

    Article  PubMed  Google Scholar 

  • Kurt G, Ergün E, Cemil B, Börcek AO, Börcek P, Gülbahar O, Ceviker N (2009) Neuroprotective effects of infliximab in experimental spinal cord injury. Surg Neurol 71:332–336

    Article  PubMed  Google Scholar 

  • Lawrence RA, Burk RF (1976) Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun 71:952–958

    Article  CAS  PubMed  Google Scholar 

  • Liu J, Qi Y, Zheng L, Cao Y, Wan L, Ye W, Fang L (2014) Xinfeng capsule improves pulmonary function in ankylosing spondylitis patients via NF-ΚB-iNOS-NO signaling pathway. J Tradit Chin Med 34:657–665

    Article  PubMed  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin-phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  • Meroni PL, Valesini G (2014) Tumour necrosis factor α antagonists in the treatment of rheumatoid arthritis: an immunological perspective. BioDrugs 28(1):S5–S13

    Article  PubMed  Google Scholar 

  • Nazıroğlu M, Kutluhan S, Ovey IS, Aykur M, Yurekli VA (2014a) Modulation of oxidative stress, apoptosis, and calcium entry in leukocytes of patients with multiple sclerosis by Hypericum perforatum. Nutr Neurosci 17:214–221

    Article  PubMed  Google Scholar 

  • Nazıroğlu M, Sahin M, Ciğ B, Aykur M, Erturan I, Ugan Y (2014b) Hypericum perforatum modulates apoptosis and calcium mobilization through voltage-gated and TRPM2 calcium channels in neutrophil of patients with Behcet’s disease. J Membr Biol 247:253–262

    Article  PubMed  Google Scholar 

  • Övey IS, Nazıroğlu M (2015) Homocysteine and cytosolic GSH depletion induce apoptosis and oxidative toxicity through cytosolic calcium overload in the hippocampus of aged mice: involvement of TRPM2 and TRPV1 channels. Neuroscience 284:225–233

    Article  PubMed  Google Scholar 

  • Pantaler E, Lückhoff A (2009) Inhibitors of TRP channels reveal stimulus-dependent differential activation of Ca2+ influx pathways in human neutrophil granulocytes. Naunyn Schmiedebergs Arch Pharmacol 380:497–507

    Article  CAS  PubMed  Google Scholar 

  • Pay S, Musabak U, Erdem H, Simsek I, Pekel A, Sengul A, Dinc A (2005) Chimerical anti-TNF-alpha, infliximab, inhibits neutrophil chemotaxis and production of reactive oxygen species by blocking the priming effect of mononuclear cells on neutrophils. Immunopharmacol Immunotoxicol 2005(27):187–198

    Article  Google Scholar 

  • Placer ZA, Cushman L, Johnson BC (1966) Estimation of products of lipid peroxidation (malonyl dialdehyde) in biological fluids. Anal Biochem 16:359–364

    Article  CAS  PubMed  Google Scholar 

  • Rudwaleit M, Listing J, Brandt J, Braun J, Sieper J (2004) Prediction of a major clinical response (BASDAI 50) to tumour necrosis factor alpha blockers in ankylosing spondylitis. Ann Rheum Dis 63:665–670

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rudwaleit M, van der Heijde D, Landewé R, Listing J, Akkoc N, Brandt J et al (2009) The development of assessment of Spondylo Arthritis International Society classification criteria for axial spondylo arthritis (part II): validation and final selection. Ann Rheum Dis 68:777–783

    Article  CAS  PubMed  Google Scholar 

  • Şahin M, Uğuz AC, Demirkan H, Nazıroğlu M (2011) Colchicine modulates oxidative stress in serum and leucocytes from remission patients with family mediterranean fever through regulation of Ca2+ release and the antioxidant system. J Membr Biol 240:55–62

    Article  PubMed  Google Scholar 

  • Santiago T, Santiago MG, Rovisco J, Duarte C, Malcata A, da Silva JA (2013) A case of infliximab-induced lupus in a patient with ankylosing spondylitis: is it safe switch to another anti-TNF-α agent? Clin Rheumatol 32:1819–1822

    Article  PubMed  Google Scholar 

  • Sedlak J, Lindsay RHC (1968) Estimation of total, protein bound and non-protein sulfhydryl groups in tissue with Ellmann’s reagent. Anal Biochem 25:192–205

    Article  CAS  PubMed  Google Scholar 

  • Túnez I, Feijóo M, Huerta G, Montilla P, Muñoz E, Ruíz A, Collantes E (2007) The effect of infliximab on oxidative stress in chronic inflammatory joint disease. Curr Med Res Opin 23:1259–1267

    Article  PubMed  Google Scholar 

  • Uğuz AC, Nazıroğlu M, Espino J, Bejarano I, González D, Rodríguez AB, Pariente JA (2009) Selenium modulates oxidative stress-induced cell apoptosis in human myeloid HL-60 cells via regulation of caspase-3, -9 and calcium influx. J Membr Biol 232:15–23

    Article  PubMed  Google Scholar 

  • Yamashita K, Takahashi A, Kobayashi S, Hirata H, Mesner PW Jr, Kaufmann SH, Yonehara S, Yamamoto K, Uchiyama T, Sasada M (1999) Caspases mediate tumor necrosis factor-alpha-induced neutrophil apoptosis and downregulation of reactive oxygen production. Blood 93:674–685

    CAS  PubMed  Google Scholar 

  • Yang Q, Zheng FP, Zhan YS, Tao J, Tan SW, Liu HL, Wu B (2013) Tumor necrosis factor-α mediates JNK activation response to intestinal ischemia-reperfusion injury. World J Gastroenterol 19:4925–4934

    Article  PubMed  PubMed Central  Google Scholar 

  • Yürüker V, Nazıroğlu M, Şenol N (2015) Reduction in traumatic brain injury-induced oxidative stress, apoptosis, and calcium entry in rat hippocampus by melatonin: possible involvement of TRPM2 channels. Metab Brain Dis 30:223–231

    Article  PubMed  Google Scholar 

  • Zou J, Rudwaleit M, Brandt J, Thiel A, Braun J, Sieper J (2003) Down-regulation of the nonspecific and antigen-specific T cell cytokine response in ankylosing spondylitis during treatment with infliximab. Arthritis Rheum 48:780–790

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The study was partially supported by Unit of Scientific Research Project, Suleyman Demirel University (Project No: BAP: 3079-TU–12). The abstract of the study will be submitted to the 6th World Congress of Oxidative Stress, Calcium Signaling and TRP Channels, held 24 and 27 May 2016 in Isparta, Turkey (www.cmos.org.tr).

Author contributions

MN and MŞ formulated the present hypothesis and MN was responsible for writing the report. YU was responsible for blood collection and patient management. MA was responsible for the analyses.

Author information

Authors and Affiliations

  1. Division of Rheumatology, Department of Internal Medicine, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey

    Yunus Ugan & Mehmet Şahin

  2. Neuroscience Research Center, Suleyman Demirel University, 32260, Isparta, Turkey

    Mustafa Nazıroğlu

  3. Department of Microbiology, Faculty of Medicine, Gaziosmanpaşa University, Tokat, Turkey

    Mustafa Nazıroğlu & Mehmet Aykur

Authors
  1. Yunus Ugan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mustafa Nazıroğlu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mehmet Şahin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mehmet Aykur
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mustafa Nazıroğlu.

Ethics declarations

Conflict of interest

The authors declare that there are no conflicts of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ugan, Y., Nazıroğlu, M., Şahin, M. et al. Anti-tumor Necrosis Factor Alpha (Infliximab) Attenuates Apoptosis, Oxidative Stress, and Calcium Ion Entry Through Modulation of Cation Channels in Neutrophils of Patients with Ankylosing Spondylitis. J Membrane Biol 249, 437–447 (2016). https://doi.org/10.1007/s00232-016-9884-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00232-016-9884-3

Keywords

Search

Navigation