Effect of IL-6 and CRP titer with antibody level on severity of COVID-19 infection

Abstract

OBJECTIVE:

SARS Coronavirus 2 (SARS-CoV-2) infection is combined with a high death rate and morbidity in different regions across the world. Interleukin-6 (IL-6) is a pleiotropic cytokine secreted in response to tissue injury, primarily produced by macrophages. C-reactive protein (CRP) is considered a part of innate immunity and is elevated in response to infection and cancer.

METHODS:

This study includes one hundred patients infected with the viral pathogen known as SARS-CoV-2 and fifty healthy individuals attending Al-Salam Hospital in Baghdad. Approximately 5 ml of samples were collected from each virus-infected patient and healthy control, then separated by centrifuge and stored in a refrigerator until testing. The study timeline was from October 1st, 2020, to January 15th, 2021. The SARS-CoV-2 (IgM, IgG) antibody was measured using the immunofluorescent technique with the Afias instrument. The IL-6 was measured using the ELISA technique with a human Elisa reader. The CRP titer was measured using the immunofluorescent technique with the Afias instrument. The level of SARS-CoV-2 (IgM, IgG) antibody was 0.01 $\pm$ 0.004, 0.02 $\pm$ 0.004, respectively, in healthy controls, while in COVID-19 patients, the level of SARS-CoV-2 IgM antibody was 2.45 $\pm$ 1.87, and the level of IgG antibody was 5.16 $\pm$ 2.63 in COVID-19 patients. The IL-6 level was 0.88 $\pm$ 0.28, 5.82 $\pm$ 3.28 in healthy controls and COVID-19 patients, respectively. The CRP titer in healthy controls was 1.25 $\pm$ 0.36, while in COVID-19 patients, it was 13.8 $\pm$ 4.85. The aim of the research is to focus on the association between IL-6 level and CRP titer, with a concentration on COVID-19 patients, and to determine if IL-6 possesses the potential to serve as a biomarker for prognosticating the extent of COVID-19 infection.

Keywords

SARS-CoV-2 anti IgM and IgG SARS-COV-2 IL-6 CRP titer inflammation immune response

1. Introduction

The Coronavirus 2 (SARS-CoV-2) is one of the intimate coronaviruses responsible for Middle East Respiratory Syndrome (MERS) as well as Acute Respiratory Syndrome (SARS) [1, 2]. It is currently accountable for the global COVID-19 pandemic, leading to a significant proportion of hospitalizations and admissions to the Intensive Care Unit (ICU) [3]. Approximately 10% of severely ill individuals require treatment for respiratory distress syndrome. The total number of leukocytes, lymphocytes, and platelets are lower than normal, with prolonged activated prothrombin time, increased C-reactive protein, and elevated muscle enzyme concentration. Lymphocytes decrease as the disease progresses. The production of cytokines such as IL-1B, IL-1RA, IL-6, IL-7, and IL-8, observed in a cytokine storm, is associated with the severity of the syndrome [4].

IL-6 is a protein known as a circulating pro-inflammatory cytokine. It is generated by T-lymphocytes, macrophages, and adipose tissue when they are stimulated. It is known to contribute to the progression of atherogenesis and is predictive of future cardiovascular morbidity and mortality [5] and malignancies [6]. The stimulation of liver cells by IL-6 initiates the acute phase response, leading to the secretion of acute phase proteins like C-reactive protein (CRP), amyloid A, haptoglobin, ferritin protein, and fibrinogen [7]. The IL-6 and IL-6R complex then binds to another receptor protein called glycoprotein 130 (gp130) and forms a dimer, initiating cellular signaling [8]. While IL-6 specifically attaches to IL-6R rather than gp130, only cells that possess both IL-6R and gp130 are capable of directly reacting to IL-6. While gp130 is present in all human body cells, IL-6R is only present in certain cell types, including hepatocytes, specific epithelial cells, and leukocytes [9]. CRP, a pentameric protein originating from the liver and classified as a pentraxin, plays a vital role in inflammatory responses. It is an essential component of the innate immune system and exhibits a rapid increase in concentration, often surpassing the standard reference range by over 1,000-fold, in response to tissue damage and infection [10]. CRP has both anti-inflammatory and pro-inflammatory properties, binding to phosphocholine, phospholipids, histone, chromatin, and fibronectin. It assists in the recognition and clearance of invading infectious agents or damaged cells. The conservative routine may activate, phagocytes cells can also be activated by Fc receiver, which speeding up cell debris removal and damaged or apoptotic cells and foreign pathogens. In a number of positions, its power intensifies tissue damage by motivating the complement organization and as a consequence inflammatory cytokine [11].

IL-6 has emerged as a potential treatment option for COVID-19-associated acute respiratory distress syndrome (ARDS) [12]. IL-6 is a multifunctional signaling protein present in various types of cells involved in immune response and tissue structure. The IL-6 receptor operates by creating a trimeric complex on the cell surface, consisting of the cytokine itself attached to the 80-kilodalton category 1 a-receptor component (known as IL-6R or CD126) and a 130-kilodalton signal transducing b-receptor glycoprotein (referred to as gp130 or CD130). The IL-6R can be found in two forms: one anchored to the cell membrane and the other soluble. Upon binding with gp130 in the presence of IL-6, it initiates signaling pathways downstream [13, 14]. Following initial inflammation, IL-6 is produced locally and then transported to the liver through circulation. This prompts the rapid activation of various acute phase proteins, including CRP, amyloid A, fibrinogen, haptoglobin, and $\alpha$ 1-antichymotrypsin [15]. Severe COVID-19 patients display higher IL-6 levels compared to stable patients. The diagnostic usefulness of IL-6 suggests its value as an early indicator of active disease [16]. This study specifically focuses on utilizing IL-6 as an assessment tool for COVID-19 severity instead of relying on CRP levels.

2. Materials and methods

The study includes one hundred patients with COVID-19 infection admitted to Al-Salam Hospital in Baghdad city, along with fifty healthy controls. Approximately 5 ml of blood was collected from each infected patient and healthy control. The samples were then separated by centrifuge and kept in a refrigerator until testing. The study period was from October 1st, 2020, to March 1st, 2021.

2.1 COVID-19 IgM and IgG antibody level

The titer of COVID-19 (IgM and IgG) antibodies was detected using the Afias instrument from Unimedica Company (Republic of Korea). The procedure was performed according to the instruction manual provided by Unimedica, with catalog number HCSCA004X.

2.2 The Il-6 level

The IL-6 level was measured by Eliza Technique (Invitrogen) from Human company (Germany) using human Eliza reader according to instruction manual of company catalog number (BMS213-2).

2.3 The CRP level

The level of CRP was detected using Afias instrument from Unimedica company (Republic of Korea) and then measured according to Unimedica instruction manual catalog no. (TTSGA21F).

2.4 Statistical analysis

Table 1
Key demographic and clinical attributes of the participants in the study

Factor	Control	Patient (M $\pm$ SD ${}^{***}$ )	$P$ value (sig $\leqslant$ 0.05)
IL-6 ${}^{*}$	0.88 $\pm$ 0.28	5.82 $\pm$ 3.28	$<$ 0.001
CRP ${}^{**}$	1.25 $\pm$ 0.36	13.8 $\pm$ 4.85	$<$ 0.001
COVID-19 IgM	0.01 $\pm$ 0.004	2.45 $\pm$ 1.87	$<$ 0.001
COVID-19 IgG	0.02 $\pm$ 0.004	5.16 $\pm$ 2.63	$<$ 0.001

${}^{*}$ IL6 $=$ Interlukin6, ${}^{**}$ CRP $=$ C-reactive protein, ${}^{***}$ M $=$ mean, SD $=$ standard deviation.

Table 2

The level of COVID-19 IgM Ab in patients (male and female) compared with healthy control

	COVID-19 IgM	$P$ value (sg $\leqslant$ 0.05)
Groups	M $\pm$ SD ${}^{*}$	Female patients	Male patients
Female control	0.01 $\pm$ 0.003	$<$ 0.001	$<$ 0.001
Male control	0.01 $\pm$ 0.004	$<$ 0.001	$<$ 0.001
Female patients	2.48 $\pm$ 1.36		$<$ 0.001
Male patients	2.42 $\pm$ 2.20

${}^{*}$ M $=$ mean, SD $=$ standard deviation.

Table 3

The level of COVID-19 IgG titer in Patients (females and males) with healthy control (females and males)

	COVID-19 IgG	$P$ value (sg $\leqslant$ 0.05)
Groups	M $\pm$ SD ${}^{*}$	Female patients	Male patients
Female control	0.01 $\pm$ 0.003	$<$ 0.001	$<$ 0.001
Male control	0.02 $\pm$ 0.004	$<$ 0.001	$<$ 0.001
Female patients	4.98 $\pm$ 2.46		$<$ 0.001
Male patients	5.35 $\pm$ 2.83

${}^{*}$ M $=$ mean, SD $=$ standard deviation.

Table 4

The level of CRP in COVID-19 patients as well as healthy control

	CRP ${}^{*}$	$P$ value (sg $\leqslant$ 0.05)
Groups	M $\pm$ SD ${}^{**}$	Female patients	Male patients
Female control	1.31 $\pm$ 0.61	$<$ 0.001	$<$ 0.001
Male control	1.18 $\pm$ 4.53	$<$ 0.001	$<$ 0.001
Female patients	12.33 $\pm$ 4.53		0.05
Male patients	15.39 $\pm$ 4.77

${}^{*}$ CRP $=$ C-reactive protein, ${}^{**}$ M $=$ mean, SD $=$ standard deviation.

Table 5

The IL-6 levels in individuals with COVID-19 compared to those in a healthy control group

	IL-6 ${}^{*}$	$P$ value (sg $\leqslant$ 0.05)
Groups	M $\pm$ SD ${}^{**}$	Female patients	Male patients
Female control	0.82 $\pm$ 0.36	$<$ 0.001	$<$ 0.001
Male control	0.93 $\pm$ 0.28	$<$ 0.001	$<$ 0.001
Female patients	4.79 $\pm$ 3.26		0.05
Male patients	6.92 $\pm$ 2.98

${}^{*}$ IL-6, ${}^{**}$ M $=$ mean, SD $=$ standard deviation.

The data analysis was performed using SPSS software, specifically “Statistical Packages for Social Sciences version 24”. ANOVA and $t$ -tests were conducted to assess differences among the studied groups. The results were presented as mean $\pm$ standard deviation (M $\pm$ SD). A $p$ -value of $\leqslant$ 0.05 was considered statistically significant.

3. Result

The SARS-CoV-2 infection has spread extensively, becoming an extreme public health challenge, known as the COVID-19 pandemic. Variations in fatality rates among countries may be attributed to irregular data recording by local health consultants. Our study included 100 patients with severe COVID-19 infection admitted to Al-Salam Hospital in Baghdad city.

Table 1 presents the baseline characteristics of the study population, indicating a highly significant association between the investigated parameters (IL6, CRP, IgM, IgG) with a $p$ -value of $\leqslant$ 0.05.

The level of COVID-19 IgM Ab titer was high in patients (female and male) with M $\pm$ SD 2.48 $\pm$ 1.36, 2.42 $\pm$ 2.20 respectively while in healthy control (female and male) with M $\pm$ SD 0.01 $\pm$ 0.003, 0.01 $\pm$ 0.004 respectively as in Table 2.

As shown in Table 3, the titer of IgG antibodies in COVID-19 patients was higher in females and males (4.98 $\pm$ 2.46, 5.35 $\pm$ 2.83) respectively, compared to the healthy controls with mean $\pm$ standard deviation values of females and males (0.01 $\pm$ 0.003, 0.02 $\pm$ 0.004) respectively.

The CRP levels in COVID-19 patients were higher in both females and males (12.33 $\pm$ 4.53, 15.39 $\pm$ 4.77) respectively compared to the median levels in the healthy control group (1.31 $\pm$ 0.61, 1.18 $\pm$ 0.45) respectively.

Table 5 was explained median of IL-6 in COVID-19 patients was high (4.79 $\pm$ 3.26, 6.92 $\pm$ 2.98) in females and males respectively while in females and males (0.82 $\pm$ 0.36, 0.93 $\pm$ 0.28) respectively.

4. Discussion

The COVID-19 disease initiated by the transmission of a novel zoonotic coronavirus called SARS-CoV-2. Its global dissemination began from its epicenter in Hubei, China. The World Health Organization officially declared it a pandemic on March 11th, 2020.

The study observed a correlation between the levels of IgM and IgG antibodies (titer) in COVID-19 patients and the levels of IL-6 and CRP. This correlation aligns with the findings of Roberto Gozalbo et al., who suggested that the rapid antibody responses to SARS-CoV-2 are associated with the inflammatory condition, as indicated by serum levels of CRP, ferritin, D-dimer, LDH, and IL-6. Consequently, we proceeded to conduct correlation analyses involving these factors [17].

CRP is a valuable biomarker for detecting infection, inflammation, and tissue damage. Its levels rise rapidly during acute inflammatory reactions [18]. On the other hand, IL-6 is an inflammatory cytokine that is particularly active during the acute phase, and higher levels in the bloodstream indicate greater lung inflammation [19]. In our current study, we assessed IL-6 levels, which were elevated in severe cases, consistent with the findings of El-Shabrawy et al., who proposed that IL-6 levels could effectively differentiate COVID-19 severity [20]. Thus, measuring IL-6 in COVID-19 patients can offer valuable predictions about the infection’s severity and prognosis at an early stage.

In our study, a correlation was observed between IL-6 and the level of IgM antibodies specific to COVID-19, as indicated in Tables 2 and 5, respectively. This correlation is also reported by Qian et al., who suggest that elevated IL-6 levels lead to increased activation of B and T cells and the presence of hypergammaglobulinemia [21]. IL-6 is recognized as a significant cytokine that is closely linked to various inflammatory diseases. Individuals infected with SARS-CoV-2 often display elevated levels of IL-6, which have been found to be correlated with the severity of symptoms, pulmonary inflammation, and extensive lung damage in patients [22]. Severe symptoms are associated with high levels of SARS-CoV-2 IgM antibodies during the active phase of infection. High levels of IL-6 in patients with COVID-19 infection determine the outcome of the infection (mortality), as mentioned by Del Valle et al, who reported that COVID-19 individuals requiring intensive care unit (ICU) admission have high levels of IL-6 [23].

In the current study, male individuals afflicted with COVID-19 infection showed IL-6 higher levers compared to females. This finding is consistent with Lotter et al, who suggested that the estrogen hormone in females has a protective role by inhibiting immune response activation [24]. This explains why the mortality rate is higher in males than in females with COVID-19 infection. Females exhibit a stronger humoral immune response compared to cellular immunity when compared with males.

When the level of IL-6 reaches the cut-off value, it can predict that patients will require mechanical ventilation, as found by Herold et al. They proposed that IL-6 levels exceeding 80 pg/ml indicate that the patient will experience respiratory failure and require mechanical ventilation [25]. IL-6 demonstrates comparable effects in the immune response to lung viral infections. It significantly enhances specific responses of adaptive immunity by activating CD8+ T cells and B cells, which are under the regulation of T regulatory cells. Additionally, IL-6 fosters the presence of neutrophils with phagocytic capabilities. However, IL-6 can also be responsible for an imbalance between Th2.

5. Conclusion

IL-6 can be used as a biomarker to predict in patients with COVID-19 the infection severity and to determine the need for mechanical ventilation. The IL-6 lever was observed higher in male patients. Additionally, the IL-6 level showed a strong association with CRP levels in SARS-CoV-2 patients.

Footnotes

Acknowledgments

Acknowledgements for Alsalam hospital staff.

Conflict of interest

None.

Ethical approval

Ethical approval for this study, including blood sample collection and patient interviews, was obtained from the Ethics Committee in the Ministry of Health/Iraq.

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