Neutrophils in Immunity and Inflammation

#### **Chapter 6**

## Neutrophil Counts and Rates in Otorhinolaryngology

*Erkan Yildiz*

#### **Abstract**

Complete blood count is a fairly inexpensive test that is widely used in the clinic. Neutrophils are also one of the most important parameters in complete blood count. They play a critical role in upper respiratory tract infection, as well as in many chronic otolaryngology diseases. It also has widespread uses in otorhinolaryngology practice. There are many publications on neutrophil counts and neutrophil lymphocyte ratios in patients. Neutrophil counts and rates play an important role in the follow-up and prognosis of many important otolaryngology diseases such as bell palsy, sudden hearing loss, allergic rhinitis, chronic otitis media, nasal polyposis, and chronic rhinosinusitis. In this chapter, the importance of neutrophils in these diseases will be discussed with the literature.

**Keywords:** neutrophil, neutrophil lymphocyte ratios, otolaryngology

#### **1. Introduction**

Neutrophils are known as basic defense cells in humans. However, recent research has shown that they do much more than defense. Neutrophil migration and its roles in inflammation gradually attracted the attention of researchers. The use of advanced technology has been effective in demonstrating the behavior of neutrophils in tissues. Neutrophils follow multiple ways to advance to the site of injury and infection [1].

Neutrophils are the most abundant leukocytes in the blood. They trigger the first damage in the host during the infection phase. They provide these roles thanks to phagocytosis, degranulation and reactive oxygen types. Overactivation of neutrophils sometimes leads to excessive tissue damage. Neutrophils causing chronic inflammation can cause loss of function in organs. When the number of neutrophils decreases, the response to inflammation decreases and this turns into immune deficiency. Neutrophils are produced in the bone marrow; this number may increase up to 1000 times in case of need.

Recently, neutrophil counts, and neutrophil lymphocyte ratios have been used in the diagnosis and follow-up of clinical errors. Numerous researches have been done on this subject and its widespread use has started in otolaryngology [2, 3].

#### **2. Neutrophils in rhinology**

In many nose-related diseases, nasal congestion is the main symptom. The most important of these diseases are allergic rhinitis, septum deviation, rhinosinusitis,

nasal polyp, and antrochoanal polyp. These diseases have chronic hypoxia and are caused by airway resistances. Hypoxia increases the mean platelet volume (**Figure 1**). Erythropoiesis develops again due to hypoxia. Therefore, change begins in hematological markers. In the study of patients with nasal septum deviation, the number of platelets decreased while the mean platelet volume increased [1]. Similarly, similar results were obtained in sleep apnea syndrome. No relation was found between laboratory values and apnea in pediatric sleep apnea children [2]. No significant difference was observed in pre- and postoperative studies in children with adenoid hypertrophy [3]. NLR has also been shown to be prognostic in patients with allergic rhinitis. Neutrophils, eosinophils, and basophils can be monitored in nasal cytology.

#### **3. Neutrophils in otology**

Neutrophils, which are effective in many areas of otolaryngology, are also important in otology. In autology, inflammatory cells increase and inflammatory changes are observed in diseases such as acute otitis media, serous otitis media, chronic suppurative otitis media, cholesteatoma, facial paralysis, sudden idiopathic sensorineural hearing loss, tinnitus, and vertigo. There are many studies on hematological markers in sudden idiopathic sensorineural hearing loss. NLR, PLR, neutrophil and lymphocyte count are known to be prognostic [4]. No relationship was found between the degree of hearing loss in the audiogram and hematological parameters [5]. Another study showed that there were laboratory parameters such as lymphocyte, lymphocyte%, platelet, mean platelet volume, platelet distribution width (MPV), neutrophil-lymphocyte ratio, platelet-lymphocyte ratio, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration [6] NLR Adult Bell While it can be used prognostically in paralysis, PLR has been reported to be meaningless [7]. In pediatric Bell palsy, it was observed that both NLR and PLR were significant [8]. In otitis media with effusion, hematological parameters were important markers determining viscosity [9]. In a study conducted with inactive patients with active chronic otitis

*Neutrophil Counts and Rates in Otorhinolaryngology DOI: http://dx.doi.org/10.5772/intechopen.93457*

media, no difference was observed in both patient groups [10]. In chronic otitis media, both NLR and PLR and MPV values are found to be high, as well as normal studies [11]. In the cholesteatoma, there is an invasive transition of the outer ear epithelium to the middle ear. During this transition, inflammatory cytokines are produced. No difference in NLR levels from control groups was found in cholesteatoma [12]. In a study of tinnitus patients, a connection was found between tinnitus and MPV values [13].

#### **4. Neutrophils in infections in the oral region**

In recurrent aphthous stomatitis, no relation was found between hematological parameters and infection. There was no difference between these patients and the control group in terms of WBC, Hb, neutrophil, lymphocyte, platelet, MPV, NLR, PLR, ESR, and CRP levels [14]. Although tonsillectomy and adenoidectomy are common surgical procedures, the effects of these operations on the immune system have not been fully established. Studies in patients with chronic tonsillitis and adenoid hypertrophy show that chronic tonsillitis and adenoid hypertrophy disrupt neutrophil chemotaxis functions and these values become normal after adenotonsillectomy. In addition, oxidant values appear to improve after this procedure [15]. It has been shown that it can be used in chronic tonsillitis patients as an effective assistant method in determining neutrophil-lymphocyte ratio, tonsillectomy, and postoperative follow-up [16]. It was stated that the mean platelet volume and neutrophil lymphocyte ratio can be used as markers in peritonsillar abscess [17].

#### **5. Neutrophils in head and neck infections**

NLR values can be used as a prognostic indicator in deep neck infections occurring after acute bacterial tonsillitis [18]. It can also be used seriously in the evaluation of chronic tonsillitis [19]. NLR values were not found statistically significant in children with obstructive sleep apnea undergoing adenoidectomy [2].

#### **6. Neutrophils in head and neck cancers**

Neutrophils play an important role in cancer formation and progression [20]. In head and neck cancers with high degree of systemic inflammation, an increase in the number of neutrophils is detected. Many studies have also been done on the prognosis of head and neck cancers. The study showed that increased neutrophil lymphocyte ratios (NLR) worsened head and neck cancer prognosis. Similar results have emerged in another study [21, 22]. In another study, a significant correlation was found between high cut-off value before treatment and poor prognosis [23]. In another study, it was determined that the rate of NLR in the nasopharyngeal carcinoma was a poor prognosis factor [24]. In patients who received pre-treatment adjuvant or primary chemotherapy in squamous cell head and neck cancers, the NLR ratio has been indicated as an important marker in demonstrating treatment success [25]. In another study related to the rates of laryngeal cancer, it was shown that the NLR rate did not change in benign lesions, premalignant or malignant laryngeal lesions of the larynx, but was prognostic in lymph node metastases. In Ref. [26], it was shown that it can help early confirmation of treatment failure in patients with metastatic HNSCC [27]. In patients with head and neck cancer of unknown P16-negative primer, NLR-6.0 was significantly associated with poor prognosis [28]. In a study in patients with oral squamous


#### **Table 1.**

*Neutrophil/lymphocyte ratio in head and neck carcinoma.*

cell cancers, increased NLR values have been shown to be a sign of poor prognosis [29]. It has higher NLR, MLR, PLR, and RDW values in children with histopathologically diagnosed lymphoma than in children with reactive LAP. Anywhere NLR, MLR, PLR, and RDW tests can be used to determine which LAP patients should be selected for biopsy (**Table 1**) [30].

#### **Author details**

Erkan Yildiz Department of Otorhinolaryngology, Afyonkarahisar Şuhut State Hospital, Şuhut/Afyonkarahisar, Turkey

\*Address all correspondence to: dr.erkanyildiz@hotmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Neutrophil Counts and Rates in Otorhinolaryngology DOI: http://dx.doi.org/10.5772/intechopen.93457*

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[9] Elbistanli MS, Koçak HE, Acipayam H, Yiǧider AP, Keskin M, Kayhan FT. The predictive value of neutrophil-lymphocyte and plateletlymphocyte ratio for the effusion viscosity in otitis media with chronic effusion. The Journal of Craniofacial Surgery. 2017;**28**:e244-e247. DOI: 10.1097/SCS.0000000000003452

[10] Tansuker HD, Eroğlu S, Yenigün A, Taşkin Ü, Oktay MF. Can serum neutrophil-to-lymphocyte ratio Be a predictive biomarker to help differentiate active chronic otitis media from inactive chronic otitis media? The Journal of Craniofacial Surgery. 2017;**28**:e260-e263. DOI: 10.1097/ SCS.0000000000003484

[11] Yigit E, Onerci Celebi O, Araz Server E, Longur ES. Neutrophil-to-lymphocyte ratio and mean platelet volume in chronic otitis media with or without cholesteatoma. Istanbul Medical Journal. 2018;**19**:162-166. DOI: 10.5152/ imj.2018.77854

[12] Klllçkaya MM, Aynali G, Tuz M, Bagcl Ö. Is There A Systemic inflammatory effect of cholesteatoma? International Archives of Otorhinolaryngology 2017;**21**:42-45. doi:10.1055/s-0036-1584363

[13] Ulusoy B, Bozdemir K, Akyol M, Mişe HI, Kutluhan A, Korkmaz MH. Investigation of neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio and mean platelet volume in patients with tinnitus. The Journal of Laryngology & Otology. 2018;**132**(2):129-132. DOI: 10.1017/S0022215117002481

[14] Karaer IC. Mean platelet volume, neutrophil-to-lymphocyte ratio, and platelet-to-lymphocyte ratio as İnflammatory markers in patients with recurrent aphthous stomatitis. Eurasian Journal of Medicine. 2020;**52**:38-40. DOI: 10.5152/eurasianjmed.2019.18486

[15] Sennaroglu L, Onerci M, Hascelik G. The effect of tonsillectomy and adenoidectomy on neutrophil chemotaxis. Laryngoscope. 1993;**103**(12):1349-1351. DOI: 10.1288/00005537-199312000-00005

[16] Yenigun A. The efficacy of tonsillectomy in chronic tonsillitis patients as demonstrated by the neutrophil-to-lymphocyte ratio. The Journal of Laryngology and Otology. 2015;**129**:386-391. DOI: 10.1017/ S0022215115000559

[17] Şentürk M, Azgın İ, Övet G, Alataş N, Ağırgöl B, Yılmaz E. O papel do volume plaquetário médio e a relação neutrófilos/linfócitos em abscesso periamigdaliano. Brazilian Journal of Otorhinolaryngology. 2016;**82**:662-667. DOI: 10.1016/j.bjorl.2015.11.018

[18] Bakshi SS. Letter to the editor regarding "predictive value of the neutrophil-to-lymphocyte ratio in patients with deep neck space infection secondary to acute bacterial tonsillitis". International Journal of Pediatric Otorhinolaryngology. 2017;**92**:193. DOI: 10.1016/j.ijporl.2015.10.028

[19] Yenigun A, Sezen S, Calim OF, Ozturan O. Evaluation of the eosinophil-to-lymphocyte ratio in

pediatric patients with allergic rhinitis. American Journal of Rhinology & Allergy. n.d.;**30**:e21-e25. DOI: 10.2500/ ajra.2016.30.4296

[20] Available from: https:// neurosurgery.directory/2019/07/07/ neutrophil-to-lymphocyte-ratio-forglioma/

[21] Takenaka Y, Oya R, Kitamiura T, Ashida N, Shimizu K, Takemura K, et al. Prognostic role of neutrophilto-lymphocyte ratio in head and neck cancer: A meta-analysis. Head & Neck. 2018;**40**:647-655. DOI: 10.1002/ hed.24986

[22] Mascarella MA, Mannard E, Silva SD, Zeitouni A. Neutrophil-tolymphocyte ratio in head and neck cancer prognosis: A systematic review and meta-analysis. Head & Neck. 2018;**40**:1091-1100. DOI: 10.1002/ hed.25075

[23] Cho JK, Kim MW, Choi IS, Moon UY, Kim MJ, Sohn I, et al. Optimal cutoff of pretreatment neutrophilto-lymphocyte ratio in head and neck cancer patients: A meta-analysis and validation study. BMC Cancer. 2018;**18**. DOI: 10.1186/s12885-018-4876-6

[24] Takenaka Y, Kitamura T, Oya R, Ashida N, Shimizu K, Takemura K, et al. Prognostic role of neutrophil– lymphocyte ratio in nasopharyngeal carcinoma: A meta-analysis. PLOS One. 2017;**12**. DOI: 10.1371/journal. pone.0181478

[25] Bojaxhiu B, Templeton AJ, Elicin O, Shelan M, Zaugg K, Walser M, et al. Relation of baseline neutrophil-tolymphocyte ratio to survival and toxicity in head and neck cancer patients treated with (chemo-) radiation. Radiation Oncology. 2018;**13**. DOI: 10.1186/s13014-018-1159-y

[26] Eskiizmir G, Uz U, Onur E, Ozyurt B, Karaca Cikrikci G, Sahin N, *Neutrophil Counts and Rates in Otorhinolaryngology DOI: http://dx.doi.org/10.5772/intechopen.93457*

et al. The evaluation of pretreatment neutrophil–lymphocyte ratio and derived neutrophil–lymphocyte ratio in patients with laryngeal neoplasms. Brazilian Journal of Otorhinolaryngology. 2019;**85**:578-587. DOI: 10.1016/j.bjorl.2018.04.013

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[30] Tezol O, Bozlu G, Sagcan F, Tuncel Daloglu F, Citak C. Value of neutrophil-to-lymphocyte ratio, monocyte-to-lymphocyte ratio, plateletto-lymphocyte ratio and red blood cell distribution width in distinguishing between reactive lymphadenopathy and lymphoma in children. Bratislava Medical Journal. 2020;**121**:287-292. DOI: 10.4149/bll\_2020\_045

#### **Chapter 7**

## Neutrophil Gelatinase-Associated Lipocalin as a Promising Biomarker in Acute Kidney Injury

*Camila Lima, Maria de Fatima Vattimo and Etienne Macedo*

#### **Abstract**

Acute kidney injury (AKI) is a common complication in several settings inside and outside hospitals. It affects millions of people around the world, and despite high levels of research funding, there is no specific treatment that changes the disease course. The basis for unfavorable outcomes related to this disease is the failure to provide early diagnosis. Currently, the diagnosis of AKI is based on serum creatinine and urine output, and both measures have several limitations, making early diagnosis difficult. In recent decades, several biomarkers of kidney injury have been proposed, with neutrophil gelatinase-associated lipocalin (NGAL) being one of most studied and promising for use in early diagnosis. Despite there being several studies on NGAL, it has not yet been applied in clinical practice; thus, furthering the understanding of the development, interpretation, and limitations of NGAL in the diagnosis of AKI is the objective of this chapter.

**Keywords:** acute kidney injury, biomarkers, neutrophil gelatinase-associated lipocalin

#### **1. Introduction**

Acute kidney injury (AKI) is a frequent complication in several clinical settings, including large surgeries [1], emergency departments [2, 3], and intensive care units (ICUs) [4]. The incidence of AKI has been increasing over the years, with about 2 million people being affected in 2010 [5], despite the efforts of researchers and organizations [6–8]. AKI is commonly followed by worse outcomes: prolonged length of ICU and hospital stay, need for dialysis, decreases in the glomerular filtration rate (GRF), development of chronic kidney disease (CKD), and increases in mortality [9–11].

In recent decades, therapeutic interventions aimed at reversing kidney dysfunction have had disappointing results in multiple settings; thus, the research focus has shifted from treatment to prevention and early detection by focusing on two main issues: diagnostic criteria and early diagnosis. In 2004, the Acute Dialysis Quality Initiative (ADQI) sought a more uniform definition of AKI, and the most recent consensus definition was published in 2012 by Kidney Disease: Improving Global Outcomes (KDIGO) [8]. The diagnosis of AKI is based on changes in serum creatinine (Scr) and urine output (UO), but neither marker is kidney specific. Efforts have been made to identify novel biomarkers that have high sensitivity and specificity.

The standardization of the diagnosis of AKI allowed us to compare the diagnoses made in different settings. However, the issue of early diagnosis is still a challenge. First, there are limitations regarding the use of Scr and UO. Second, determining the need for renal replacement therapy is difficult due to a lack of information about whether the AKI is transient or persistent. Last, the research advances identifying early biomarkers have thus far been inaccessible in clinical practice [12].

Neutrophil gelatinase-associated lipocalin (NGAL) [13] has been far and away the most promising biomarker to help fill this gap, and its diagnostic capabilities as a biomarker have been confirmed in a large number of clinical trials. This chapter aims to present information on the role of NGAL in the renal injury process, its expression in the kidney, confounding factors, the type of assay used, whether plasma or urine NGAL has better accuracy, the cutoff values in normal individuals, the accuracy of NGAL for diagnosing AKI, and the evaluation of other outcomes.

#### **2. The role of NGAL**

In 1993, Kjeldsen et al. [13] isolated lipocalin as a protease-resistant polypeptide covalently bound to neutrophil gelatinase, named neutrophil gelatinase-associated lipocalin (NGAL), also known as siderocalin, lipocalin 2 or oncogene 24p [14].

NGAL is a 25 kilodalton (kDa) protein covalently bound to gelatinase in neutrophil-specific granules. NGAL is expressed at very low levels in various human tissues, including the kidneys, uterus, prostate, salivary gland, trachea, lungs, stomach, and adult and fetal colon [15, 16]. The anti-inflammatory function of NGAL is demonstrated by increased NGAL expression in proliferative epithelia, inflammatory areas, and intestinal malignancies [17].

In normal kidneys, the expression of NGAL is mainly released by the thick ascending limb and the intercalated cells of the thick collecting duct. Some NGAL expression is also present in the proximal tubular epithelium, once NGAL is filtered by the glomerulus and reabsorbed by the proximal tubule in a megalin-dependent manner [17, 18]. The physiological function of NGAL in the kidneys is unknown; however, the role of NGAL in renal morphogenesis is under consideration [19]. NGAL also has a predominant role in the regulation of cell proliferation, repair processes, and tubular reepithelization. NGAL expression corresponds to an additional iron transport pathway, which increases the transcription of hemeoxygenase, an enzyme with proliferative and antiapoptotic effects that protects and preserves proximal tubular cells [20, 21].

Several biological functions for NGAL have been suggested; in the kidney, NGAL release is associated with ischemic or nephrotoxic insults. Additionally, a decrease in tubular reabsorption after AKI may lead to a further increase in urinary NGAL concentration, resulting acquiring a status of the "troponin" of the kidneys [22–24].

*KEY POINT: The role of NGAL remains unclear, but its release mainly from the distal tubule has been associated with an increase in kidney injury.*

#### **3. Confounding factors affecting NGAL**

The conditions that can interfere with the performance, sensitivity, and specificity of NGAL, already identified as a biomarker, are sepsis, chronic obstructive pulmonary disease, and cardiac dysfunction, and the presence of these conditions may act as confounding factors for NGAL measurements. The predictive performance of NGAL seems to also be influenced by age (higher predictive value *Neutrophil Gelatinase-Associated Lipocalin as a Promising Biomarker in Acute Kidney Injury DOI: http://dx.doi.org/10.5772/intechopen.93650*

in children than in older patients), sex (higher predictive value in female patients than in male patients), urinary tract infection, and impaired renal function (higher predictive value in patients with chronic kidney disease) [25–27].

Sepsis will be more thoroughly addressed in the next chapter.

*KEY POINT. Controlling for confounding factors in clinical trials is vital to maintain the internal validity of a study.*

#### **4. Types of NGAL assays**

The commercialization of NGAL as the gold standard for the diagnosis of AKI is somewhat controversial [28]. There are many types of NGAL assays available on the market that use different nonautomated ELISA platforms, which makes it difficult for comparisons to be made among studies from around the world.

The first kit for the quantitative and automated determination of NGAL by the ELISA method was developed by Abbott Laboratories (Abbott Park, IL, USA) for the urinary evaluation of NGAL, with a cutoff value of 141 μg/L (95% CI 125–158 μg/L). An EDTA plasma blood test was created by the Triage Meter platform (Biosite-Inverness Medical, Waltham, USA) with a cutoff value of 163 μ/L (CI 109–221 μ/L) [29].

Bioporto (Bioporto Diagnostics A/S, Gentfte, Denmark) developed a new particleenhanced turbidimetric immunoassay (PETIA) that has the advantages of flexibility (adaptation for clinical use in different analyzers), automation (closer to clinical practice), and applicability in different biological matrices (urine and plasma) [30].

The chemiluminescence test is an alternative to assess NGAL, and it is commonly used to analyze studies with small animals since it is possible to do the analysis with few substrates [31].

Because there are three known molecular forms of NGAL, the assay of interest should differentiate the 25 kDa NGAL monomer produced by the monocyte tubular epithelial cells from other forms of NGAL: 45 kDa NGAL, from the homodimer predominantly secreted by neutrophils, and the 145 kDa NGAL/matrix metalloproteinase-9 (MMP9) covalently complexed heterodimer [32, 33].

According to Mårtensson et al. [34] and Cai et al. [33], the combination of two ELISAs, may improve the diagnostic accuracy of NGAL, one to determine the monomeric form and the other to determine the homodimeric form.

The confounding factor of sepsis is described herein and is dependent on the assay method, as well as whether the chosen kit is less sensitive to 25 kDa NGAL expressed by tubular epithelial cells. The test can also measure the 45 kDa homodimer predominantly secreted by neutrophils, which are common in sepsis, and the increase in neutrophils increases homodimeric NGAL expression and results in false positives.

*KEY POINT: Choose a method closer to those used in clinical practice and a test more accurate for measuring the monomeric form of NGAL expressed by tubular epithelial cells after injury.*

#### **5. Plasma or urine NGAL measurement and normalization of urine values**

The consensus is clear that NGAL measured in urine has better performance [35], because the release and increase in NGAL will occur first in urine. However, the collection of urine depends on the urine output, which is sometimes not available. Some benefits of plasma NGAL are that it is available at any time and is more accurate in anuric or oliguric patients.

The issue about the normalization of urinary NGAL by correction for the urinary creatinine level is debatable, but it has been used to correct for urine output in cases of oliguria or pollakiuria, avoiding inaccurate concentration or dilution measurements of the biomarkers. The fact is that the creatinine release time is different from the biomarker release time, and the normalized level will be affected by this difference and will not represent a real physiological value [36].

*KEY POINT: Urinary NGAL is released earlier than plasma NGAL, and the accuracy of the normalization of urinary NGAL by creatinine is debatable.*

#### **6. Cutoff values of NGAL in normal individuals**

The determination of normal NGAL levels in healthy adults has been inadequately described in the literature. However, some studies have been performed, such as that of Cullen et al. [26], which analyzed urine by the Abbot-Architec assay in 174 healthy people (100 men and 74 women aged between 19 and 88 years). The value of the immunoassay result was normalized by urinary creatinine, and the cut-off value was 107 μ/mmol (13 μ/mmol). There was a higher concentration of creatinine-normalized urinary NGAL in women, in the elderly and in patients with leukocyturia.

The study reported by Stejkal et al. [37] analyzed BioVendor's NGAL assay using the serum of 136 healthy, nonobese individuals (53 men and 83 women). The authors reported median NGAL values (78.8 μg/L for men and 80 μg/L for women). Pernnemans et al. [27] analyzed the NGAL ELISA (RD System Europe, Abingdon, UK) and other urinary biomarkers in 338 healthy individuals (199 women and 139 men, aged 0 to 95 years). They reported that the NGAL reference range of the 21–95 years age group was 73.88–211.16 μg/L in women and 149.26–182.58 μg/L in men, and there was higher expression in elderly individuals.

An NGAL PETIA (Bioporto Diagnostics A/S, Gentfte, Denmark) [36] was evaluated for 200 healthy nonobese individuals (137 men and 63 women, with a mean age of 39 years (SD 11.2)). They proposed reference plasma concentrations from 38.7–157.6 ng/ml for women and 24.4–142.5 ng/ml for men and proposed reference urine concentrations of <9–54.5 ng/ml for both sexes. The authors reported that the mean values in men were higher than in women, 78.9 ng/ml vs. 73.8 ng/ml, respectively; there was a significant difference in NGAL in relation to age.

In addition to the variability of the chosen immunoassay, the unit of measurement in the interpretation of the NGAL studies should be considered—the most commonly found are ng/ml, μg/L, ng/dl, mg/ml and μg/mmol.

*KEY POINT: The median cutoff value for urinary NGAL in healthy men was between 78.8 and 182.58 μg/L, and the median plasma value was between 24.4 and 142.5 ng/ml in men. The broad variability of the results difficult to interpret.*

#### **7. NGAL to predict AKI**

NGAL is the most widely investigated AKI biomarker. Its performance for predicting AKI has been evaluated in various settings, such as in pediatric and adult cardiac surgery patients, in critically ill patients, and in patients in the emergency room, as well in kidney transplant and other settings [38, 39].

Numerous studies have demonstrated the ability of NGAL to diagnose AKI. For example, the study reported by Constantin et al. [40], that evaluated the plasma NGAL of 88 patients at ICU admission, found a sensitivity of 82%, specificity of 97% and AUC of 0.92 to cut-off value of 155 mmol/L to predictor of AKI.

*Neutrophil Gelatinase-Associated Lipocalin as a Promising Biomarker in Acute Kidney Injury DOI: http://dx.doi.org/10.5772/intechopen.93650*

A multicenter study, reported by Di Somma et al. [41], with 665 patients admitted to the emergency department, assessed plasma NGAL in several points after admission. Serial evaluation of NGAL at times zero and six hours provided a high negative predictive value (NPV) (98%) to rule out the diagnosis of AKI within six hours of the arrival of patients to the emergency department. The NGAL value at admission could demonstrated a strong predictive value for in-hospital mortality of the patient, with a cut-off value of 400 ng/ml.

In the meta-analysis by Haase et al. [42]—with 19 studies, totaling 2538 patients, of whom 487 (19.2%) developed AKI—NGAL was demonstrated to have diagnostic and prognostic value for AKI, with an OR of 18.6 (95% CI 9–38.1) and AUC of 0.81 (95% CI 0.73–0.89). The cut-off value ranged from 100 to 270 ng/ml, but a value of 150 ng/dl was suggested for the diagnosis of AKI.

In another recent meta-analysis by Zhou et al. [39]—with 24 studies, a total of 4066 patients from 9 countries, including studies with serum and urinary NGAL the sensitivity for the diagnosis of AKI was 0.68 (95% CI, 65–0.70), and the specificity was 0.79 (95% CI 0.77–0.80).

In the study by Singer et al. [38], urinary NGAL was useful for classifying and stratifying patients with established AKI: the level of NGAL>104 μg/L indicated intrinsic AKI (odds ratio of 5.97), while the level of NGAL <47 μg/L indicated unlikely intrinsic AKI (odds ratio of 0.2). In the logistic regression analysis, NGAL was able to predict the worsening of the RIFLE class, the need for RRT and inhospital mortality. The performance of NGAL to evaluate other outcomes will be discussed in the next chapter.

*KEY POINT: Despite the good results of NGAL for predicting AKI, the variability of the cutoff value is still a challenge for applying NGAL in clinical practice.*

#### **8. The early timing diagnosis by NGAL versus standard serum creatinine**

The study by Bennett et al. [43] clearly indicated that urinary NGAL is a powerful early biomarker of AKI after cardiopulmonary bypass that preceded the increase in serum creatinine by 2–3 days. Studies have shown that elevation of NGAL is detectable after 3 hours and peaks approximately 6–12 hours after injury. The elevation can persist up to 5 days according to the severity of injury [44–46]. In addition to Benett's study, other studies in general have failed to reach conclusions about the early timing diagnosis of NGAL, which is the main finding required to reach a therapeutic window and better evaluate future medication targets in AKI.

*KEY POINT: If you perform a study or analyze a biomarker, remember to compare the pattern of biomarker early timing diagnosis with serum creatinine.*

#### **9. Evaluation of other outcomes by NGAL**

Several studies have assessed the diagnostic value of NGAL to predict AKI, but only a few have analyzed the early diagnosis in hours/days and compared it with serum creatinine, as seen in the last chapter. Still fewer studies have evaluated the predictive performance of NGAL in other outcomes, such as the need for RRT, recovery of renal function, progression to end stage renal disease (ESRD) and mortality, which will be discussed in this chapter.

In the meta-analysis by Hall et al. [47], for 91 kidney transplant patients, the incidence of need for RRT was 4.3%, and NGAL, in this scenario, had an OR of 12.9 and AUC of 0.78. In the same study, NGAL and urinary IL18 were predictors of the need for RRT up to 1 week after transplantation. NGAL presented a good AUC of

0.81 (95% CI 0.70–0.92) 6 hours after transplantation and was also a predictor of graft recovery for up to 3 months.

In the study conducted by Constantin et al. [40], the cutoff value of NGAL to assess the need for RRT was 330 mmol/L. The value of urinary NGAL (Architect, Abbot Park, IL) was correlated with the need for dialysis (r: 0.48 P: 0.01), presenting an AUC of 0.86 2 hours after cardiopulmonary bypass in children [43].

A recent meta-analysis by Klein et al. analyzed 12 studies to predict the need for RRT and found an AUC of 0.70 (95% CI 0.63–0.80) for NGAL [48].

Bhavsar et al. [49] concluded that higher levels of NGAL (measured by the Luminex assay) were associated with stage 3 CKD incidence. Some researchers have discussed whether the association of NGAL level is not exclusively related to the increase in neutrophils already described by Tian et al. [50] and maintain that further studies would be needed to elucidate this issue.

In the meta-analysis by Haase et al. [42], the incidence of mortality was 5.4%, and NGAL, in this scenario, showed an OR of 8.8 and AUC of 0.70.

In the Ariza study [51], PNGAL and UNGAL were demonstrated to be strong predictors of prognosis, and UNGAL was significantly predictive of the MELD score using the 28-day mortality score AUC of 0.88 (0.83–0.92).

In the study by Bennett et al. [43], the value of urinary NGAL (Architect, Abbot Park, IL) was also correlated with mortality (r: 0.53 p 0.01), with an AUC of 0.91, 2 hours after cardiopulmonary bypass in children.

The study by Dent et al. [52], using PNGAL (Biosite Inc., San Diego, USA) in 120 children undergoing cardiopulmonary bypass (CBP) and a cut-off value of 150 ng/ml and AKI prediction, found an AUC of 0.96 2 hours after CBP. PNGAL was also strongly correlated with the duration of AKI (r = 0.57, p < 0.001) and hospital stay time (r = 0.44, p < 0.001), and PNGAL at 12 hours was correlated with mortality (r = 0.48, p: 0.004).

In the study by Daniels et al. [53], PNGAL (Alere Inc., Waltham, USA) was measured in 1393 adult patients with cardiovascular disease (CVD) who were followed for 11 years. Of these, 436 did not survive, and 169 died from CVD. PNGAL was a predictor of CVD mortality, with a risk ratio of 1.33% and a risk ratio of 1.19% for all causes of mortality.

*KEY POINT: Evaluating outcomes by NGAL beyond the limitation of only the diagnosis of AKI is important to know how more than one parameter evaluates the outcome and prognosis, and it could help physicians by indicating an early need for RRT, for example.*

#### **10. Conclusion**

This brief review, based on accumulated evidence, discussed the role and value of NGAL in the diagnosis and prognosis of AKI. Studies' findings suggest that induction of NGAL plays an important role in kidney function preservation, reducing apoptosis, and enhancing proliferative responses. In kidney injury, rapid and massive upregulated synthesis of NGAL occurs in the distal tubule, which quickly increases the concentration of NGAL in urine [54]. In addition, other important considerations have been provided as "key point" to help researchers move the NGAL analysis to clinical practice as soon possible.

#### **Conflict of interest**

The authors declare that there are no conflicts of interest regarding the publication of this chapter.

*Neutrophil Gelatinase-Associated Lipocalin as a Promising Biomarker in Acute Kidney Injury DOI: http://dx.doi.org/10.5772/intechopen.93650*

#### **Author details**

Camila Lima1 \* † , Maria de Fatima Vattimo2‡ and Etienne Macedo1,3§

1 Internal Medicine Department, Nephrology Division, University of Sao Paulo, Sao Paulo, Brazil

2 Medical Surgical Department, School of Nursing, University of Sao Paulo, Sao Paulo, Brazil

3 Department of Medicine, Nephrology Division, University of California, San Diego, USA

\*Address all correspondence to: camilaxlima@gmail.com

† Current Address: 455 Av. Arnaldo Dr, 01246-903, Cerqueira Cesar, Sao Paulo, Brazil.

‡ Current Address: 419 Av Dr Eneas de Carvalho Aguiar, Cerqueira Cesar, Sao Paulo, Brazil.

§ Current Address: 9500 Gilman Dr, MC 0892, La Jolla, California, United States of America.

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 8**

## The Role of Neutrophil Extracellular Traps (NETs) in the Pathogenesis and Complications of Malignant Diseases

*Sheniz Yuzeir and Liana Gercheva*

#### **Abstract**

It was recently proved that neutrophils and platelets are active participants in some inflammatory processes as well as a number of pathological conditions, including neoplastic diseases and thrombosis. It has been found that circulating neutrophils actively affect the mechanisms of tumour genesis, and along with platelets, act as independent regulators of different complications in infectious and malignant diseases. A few years ago, it was found that neutrophils have the ability to release extracellular traps (called neutrophil extracellular traps or NETs). Thus, neutrophils use both intracellular and extracellular mechanisms to limit inflammatory complications. Several recent studies confirmed that NETs increase considerably in malignant diseases, demonstrating that tumour-induced NETosis is a clinically significant process. It is recognised as an element of tumour biology, as it participates in tumour progression and angiogenesis. Neutrophils and the NETs released from them are stimulators of thrombotic processes in physiological and pathological conditions. Several reports demonstrate the connection between NETs and thrombosis. The presence of NETosis serves as a potential risk factor for thrombotic complications in malignant diseases. This chapter summarises the current knowledge of NETosis and the mechanisms that lead to the formation of NETs, including the role of circulating platelet–neutrophil complexes as regulators of tumour-induced NETosis in malignant diseases.

**Keywords:** NETosis, neutrophils, platelets, malignant diseases, infections

#### **1. Introduction**

One of the important causes of increased mortality in patients suffering from different inflammatory and neoplastic diseases is thrombosis of a large artery or vein. Recently, the attention of study groups has been drawn to the newly discovered functions of neutrophils, which confirm their significant role in not only inflammatory processes but also a number of pathological conditions, including neoplastic diseases and thrombosis [1, 2].

Neutrophils, as inherent mediators of immune defence, play an important role in various inflammatory processes [1]. Studies of the content of neutrophil granules reveal that an abundance of enzymes and macromolecules have roles in the different cellular interactions of granulocytes [3]. Enzyme-rich content reflects the active participation of neutrophils in the protective inflammatory response to bacteria, fungi and, to a lesser extent, other infections. These enzyme-mediated reactions are activated after membrane signalisation from the granulocyte plasmalemma, which possesses adhesive proteins, receptor molecules and ionic channels with pump mechanisms [4, 5]. Some receptor molecules have enzyme activity that inactivates cytokines and interleukins or activates intracellular processes for chemotactic movement of neutrophils through circulation from endothelial pores to tissues [6, 7]. Their ability to absorb pathogens and activate apoptosis through intracellular phagolysosomes is well known. After apoptosis was first described in 1972, several other mechanisms of cellular death were revealed, and decoding the paths leading to cellular death has remained a subject of discussion. In 2004, a group of scientists led by Brinkman [8] proved that stimulation of neutrophils with interleukin 8 (IL-8) or lipopolysaccharides (LPSs) causes liberation of chromatin in the extracellular space. Thus, these specific inflammatory cells release neutrophilic extracellular traps (NETs), which are formed mainly by decondensed nucleosomes and chromatin extracted from intracellular granules, such as neutrophil elastase and myeloperoxidase. So, it has been proven that neutrophils use both intracellular and extracellular mechanisms to confine infections.

#### **2. Neutrophil Extracellular Traps (NETs)**

The process of NET formation is called NETosis. This is characterised as a new process of cellular death that leads to chromatin decondensation, followed by cellular protein disintegration, lysis of the cytoplasm membrane and release of NETs [9]. The process of NETosis is dependent on enzyme peptidylarginine deaminase 4 (PAD4), which catalyses the transformation of histone-associated arginine residues into citrulline. It is mediated through the transformation of the α-amino group of arginine into a ketone group with subsequent chromatin decondensation [10–12].

The molecular mechanisms leading to NET formation remain unclear. According to recent data, the release of NETs in the extracellular space depends on two important processes: generation of reactive oxygen species (ROS) and decondensation of chromatin. The production of ROS is realised by activating nicotinamide adenine dinucleotide phosphate, (NADPH) oxydase as well as by including additional signal paths that mediate different forms of NETosis. The activation of protein kinase C leads to the assembly of a NOX 2 complex in the phagosome membrane and subsequent electronic transport and formation of hydrogen peroxide (H2O2), which is a powerful inductor for the generation of NETs [9, 13].

The second process leading to the formation of NETs is decondensation of chromatin. Interestingly, the additional release of lipopolysaccharides demonstrates that neutrophil elastase from azurophilic granules is the etiologic agent of this phenomenon. After neutrophil activation, the enzyme moves to the cellular nucleus and decomposes histones, particularly histone 4, while the nuclear changes and decondensation of chromatin are both proportional to the level of histone 4 decomposition [14]. This implies that the other enzyme included in the process of NET formation is myeloperoxidase [15]. An important step in NET formation is epigenetic modification of the histones, so-called histone citrullination through activation of the enzyme peptidyl-arginine deaminase (PAD4). Histone citrullination prevents histone methylation and further transcription, eventually resulting in chromatin decondensation [12, 16]. Releasing NET into the extracellular space involves a series of interconnected processes. The first process occurs

#### *The Role of Neutrophil Extracellular Traps (NETs) in the Pathogenesis and Complications… DOI: http://dx.doi.org/10.5772/intechopen.93651*

when the nuclear and granular membrane disintegrates and elastase enters the nucleus; the second process involves the hypercytrulinisation of histones; the third process includes decondensation in the cytoplasm; and the fourth process transpires when the plasma membrane ruptures and nuclear material is extruded from the cell in the outer space. Certain enzymes, such as peptidyl arginine deminase type IV (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO), play key roles in the chromatin decondensation process [17, 18]. Extracellular DNA, histones and granular enzymes form a network of NETs that capture endogenous (e.g. platelets) and external (e.g. bacteria) particles. In addition, molecules are involved in the formation of HETs. Negatively charged DNA has been determined to act as the basis for NET and to interact with other NET components through a positive electrostatic charge. Although a number of studies have used PMA as an inducer of NET, the exact intracellular pathway that leads to the release of NET has yet to be determined [19, 20].

Most studies have concluded that an important autophagy process is activated in the formation of NETs. Autophagy is an anti-apoptotic mechanism that activates in response to cell stress. It occurs in order to regulate protein and organelle turnover, ensuring cell survival [21]. The protein kinase mammalian target of rapamycin (mTOR) negatively regulates autophagy, which is also involved in the formation of NET [22, 23]. Most studies have indicated that an important autophagy process is activated during the formation of NETs. Some intracellular signalling pathways, such as the PICK3 blocking autophagy, inhibit the release of NETS. It has been presumed that autophagy is critical to the release of NET in both infectious and non-infectious diseases, such as sepsis, familiar Mediterranean fever (FMF), gout and inflammatory-driven fibrosis [24, 25].

The main components of NETs are DNA, histones and proteases, which have pro-coagulation properties. Histones have a marked cytotoxic effect on the vascular endothelium and can induce thrombosis [26]. Sulphurous proteases, such as neutrophil elastase, inactivate the tissue factor pathway inhibitor and lead to hypercoagulation and fibrin deposition [27].

The critical role of neutrophils in the processes of tumour genesis has been emphasised in a number of publications. As inflammatory cells, they release different types of cytokines and chemokines that, through activation of intercellular interactions and modulation of the immune response, influence the tumour microenvironment [28]. In addition, the proteases secreted by neutrophils have a specific role in regulation of the proliferation of tumour cells, tumour angiogenesis and the metastasis process. Different activating cytokines [IL-8, granulocyte colony-stimulating factor (G-CSF) and tumour necrosis factor alpha], myeloperoxidase, neutrophil elastase and histone citrullination are included in the NET release process [21].

Some studies emphasise the critical role of G-CSF in tumour-induced NETosis. It is known that a major proportion of tumour cells produce G-CSF, which induces neutrophilia, a common finding in malignant diseases, which is usually related to poor prognosis. By releasing NETs, neutrophils provide a scaffold and stimulate the processes of platelet adhesion and aggregation. They are closely linked to tumour cells in vivo and in the tumour vasculature, but their role in tumour biology is still a subject of discussion.

Tumour-induced neutrophils have both pro- and anti-tumour potential. On the one hand, they secrete cytokines, generate thrombin and initiate positive feedback for stimulation of tumour growth, tumour invasion and maintenance of tumour angiogenesis. On the other hand, the anti-tumour potential of neutrophils is explained by their direct cytotoxic interaction with cancer cells, which stimulates the apoptotic decomposition of tumour cells due to their antibody-dependent cell-mediated cytotoxicity and favours their migration [29]. A number of studies

have confirmed that tumours expressing high levels of G-CSF are powerful inductors of NETosis [30]. Also, cytokine IL-8, which is frequently expressed by different tumour cells, is described as a NET-inducing factor and has recently been proven to be essential for tumour-induced NETosis [31].

NETosis occurs in some infectious diseases as well as a number of non-infectious diseases. Some years ago, Hakkim and colleagues demonstrated that its incidence has increased in autoimmune diseases, such as lupus erythematodes [32]. Also, NETosis has recently been described to have a role in diabetes. It has been proven that hyperglycaemia is the cause of more frequent neutrophil activation and NET formation [33]. Additionally, it is assumed that NETs are included in the pathogenesis of some conditions, such as atherosclerosis [34].

The neutrophils and NETs released from them are important stimulators of thrombus formation processes in individuals with physiological and pathological conditions. Malignant diseases are risk factors for different types of thrombosis, and most often this is associated with the process of hypercoagulation as well as with increases in the capacity of activated neutrophils to form NETs. In the nineteenth century, Armand Trousseau reported the first data confirming the association of cancer with thrombosis, later called Trousseau's syndrome. The connection between NETs and thrombosis was demonstrated later, when Fuchs and colleagues showed that neutrophil extracellular traps provide a scaffold for activation of circulating platelets [35]. Since then, NETs have been considered to be involved in thrombosis processes related to cancer, and NETosis has been suggested to be a potential target for preventing thrombotic complications in malignant diseases [36]. Higher levels of NETs in blood stimulate the development of both arterial and venous thrombi.

Tumour-induced platelets have a critical role in the process of NETs' release. Their immunomodulating effects are partially connected to their interactions with the inherent mediators of the immune system. The hyperactive condition of platelets in individuals with malignant diseases is due to the fact that many tumours express a tissue factor that leads to fibrin formation and platelet activation [37]. Their increased activation leads to the development of thrombosis and influences tumour genesis [38]. Therefore, it is generally accepted that neutrophils and platelets are important regulators of tumour-induced NETosis.

It is established that the number of activated neutrophils and platelets increases in the presence of inflammatory and neoplastic diseases. The formation of complexes between these cells is the main mechanism that connects haemostasis with inflammatory processes [39, 40]. About 50 years ago, the phenomenon of platelets adhering to neutrophils was described and termed 'platelet satellitism' [41]. These complexes are observed in a number of pathological conditions, such as bronchial asthma, chronic ulcerative colitis, sepsis, rheumatoid arthritis and acute coronary syndrome [42–45]. The first time these interactions were observed specifically in patients with cancer of the prostate gland was in 1975 [46]. The platelet–neutrophil complexes that were formed led to the mutual activation of platelets and neutrophils as well as the release of cytokines, exposition of adhesion molecules and receptors on the cell surface [39, 40]. This process of complex interaction is realised between the adhesion molecule P-selectin (CD62 P), which is located on the platelet surface, and the ligand P-selectin glycoprotein ligand-1 (PSGL-1), which is situated on neutrophils [47]. The important role of the interactions between integrated receptor molecules, such as glycoprotein 1b-IX-V and glycoprotein IIB/ IIa on the platelets and alpha-M-beta-2 on neutrophils, which initiate intracellular signal transduction [48–51], is emphasised. A number of studies demonstrated that activated neutrophils cause activation of platelets, similar to the way that activated platelets stimulate greater synthesis of NETs. Some interesting facts show that these

#### *The Role of Neutrophil Extracellular Traps (NETs) in the Pathogenesis and Complications… DOI: http://dx.doi.org/10.5772/intechopen.93651*

circulating platelet–neutrophil complexes form the so-called 'metastatic niche' and accelerate the process of metastasis formation [52]. Tumour-induced NETosis is a promoter of subsequent pathological processes connected to the development and progression of cancer [53].

Some prospective studies cite data confirming increased levels of circulating platelet–neutrophil complexes in patients with myeloproliferative diseases. The correlation of these complexes with the stage of the disease, the clinical course and treatment is of great interest. It has been found that patients with advanced stages of disease have higher levels of circulating complexes in their blood. In addition, the process of neutrophil activation, which is characterised by increased membrane expression of CD11b, release of proteolytic enzymes and platelet–neutrophil aggregates, contributes to the development of thrombosis [54].

Chemotherapy is related to an increased risk of developing thrombosis, but the pathogenetic mechanisms and the cytostatic agents that modulate haemostasis have not been fully clarified [55]. It is known that some of the cytostatics (e.g. doxorubicin, epirubicin) used in therapy for malignant haemopathies and solid tumours induce tissue factor (TF) expression on the cancer cells, monocytes and the vascular smooth muscle fibres [56, 57]. Global coagulation assays are used to examine the effects of chemotherapeutic agents on the haemostatic balance, providing a good assessment of the pro- and anti-coagulant activity of these cells. Interestingly, treating patients with doxorubicin and epirubicin stimulates the expression of tissue factor and increases thrombin generation in defibrinated plasma. The procoagulant effect of anthracyclines on endothelial cells can cause an increase in the exposure of phosphatidylserine by caspase activation. Their effects on the activation of protein C have also been studied [58, 59]. To summarise, studies performed in vitro suggest that doxorubicin and epirubicin have the greatest prothrombotic potential to induce a procoagulant phenotype, as they provoke both apoptosis and NETosis.

In most publications, the preferred method to evaluate and assess the levels of circulating platelet–neutrophil complexes is flow cytometric analysis of venous blood after stimulation of the complexes with adenosine diphosphate and phorbol 12-myristate 13-acetate. The conjugated antibody CD62 P (P-selectin) is used to assess the expression of CD11b and platelet–neutrophil complexes (CD41, CD45). Using this method, it has been established that the higher the percentage of circulating complexes in blood, the higher the risk of thrombotic complications [60].

In conclusion, it should be emphasised that neutrophils and platelets are key regulators of tumour-induced NETosis. The neutrophils and formed NETs are important stimulators of the thrombotic processes. The identification of NETs and the characterisation of their role in disease have revived the overlooked role of neutrophils in disease pathogenesis. The analysis and evaluation of the levels of the circulating platelet–neutrophil complexes in blood in neoplastic diseases can be used as potential predictors of the occurrence of thrombosis. The flow cytometric method used for evaluation of the interaction between neutrophils and platelets achieves accurate and reproducible results.

*Inflammation in the 21st Century*

#### **Author details**

Sheniz Yuzeir\* and Liana Gercheva Clinic of Hematology, St. Marina University Hospital, Medical University of Varna, Varna, Bulgaria

\*Address all correspondence to: shenizyuzeir@abv.bg

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Role of Neutrophil Extracellular Traps (NETs) in the Pathogenesis and Complications… DOI: http://dx.doi.org/10.5772/intechopen.93651*

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Section 4
