IL-4 Serum Level Estimation in Myeloproliferative Neoplasm Patients

Back Ground: Myeloproliferative neoplasm (MPN) is a long-term blood disease that has an excess production of mature hematopoietic pluripotent stem cells in the bone marrow. In the early fifties, W. Dameshek structured the Myeloproliferative disorders that are at present the World Health Organization (WHO) changed it to Myeloproliferative Neoplasms (MPNs). According to the Iraqi cancer registry, Chronic Myeloproliferative disorders in the male is 0.62% and the incidence rate is 0.36, in female Chronic Myeloproliferative disorders (45 case) is 0.31% and the incidence rate is 0.24. The JAK2-V617F genetic mutation is approximately seventy percent of the Myeloproliferative Neoplasm cases. Interleukin-4 plasma and serum levels are significantly increased in MPNs different types. Objectives: The goal of this study is to estimate the IL-4 serum levels in the JAK2-V617F negative and positive mutation in the Iraqi MPNs patients. Materials and Methods: Total of (60) patients screened by cohort prospective study of having MPN who are patients presented to the National Center of Hematology / Al-Mustansiriyah University. Depending on the JAK2-V617F genetic mutation we classified our MPNs cases into 3 groups: JAK2-V617F negative (N: 20), JAK2-V617F positive (N: 40) and control group (10). Blood sample (5) ml was obtained from each individual in each group, by venipuncture using disposable syringes for IL-4 serum estimation by Enzyme Linked ImmunoSorbent Assay (ELISA) technique. Results: A clear indication of significant differences was observed between IL-4 serum level in JAK2-V617F negative samples and control samples (P < 0.05). Conclusion: The IL-4 serum level is high in MPNs patients, which is one of the immune evading mechanisms of the cancerous acting to imbalance the Th1/Th2 ratio and enhancing the anti-apoptotic activity inside those cells.


Introduction
Myeloproliferative neoplasm (MPN) is a long-term blood disease that has an excess production of mature hematopoietic pluripotent stem cells in the bone marrow. In MPN, there is unusual increase in the output of a specific cell kind. So, MPN includes an incorrect equilibrium in the output of various hematocytes kinds, also unusual output of any given blood cell kind (1). In the early fifties, W. Dameshek structured the Myeloproliferative disorders that are at present the World Health Organization (WHO) changed it to Myeloproliferative Neoplasms (MPNs). Philadelphia chromosome positive -Chronic myeloid leukemia (CML) and 3 Philadelphia chromosome negative: Primary Myelofibrosis (PMF), Essential Thrombocythemia (ET), and Polycythemia Vera (PV) are 4 classical kinds of Myeloproliferative Neoplasms (2).
In US, It is more common in males that suffer from 2.4 new cases / 100,000 while in females it's about 1.4 new cases / 100,000. The 5-year survival rate of MPNs patients is approximately 67.6%, and the fatality median age is approximately seventy seven years (3). According to Iraqi cancer registry, Chronic Myeloproliferative disorders in male is 0.62% with incidence rate of 0.36, while, in female the Chronic Myeloproliferative disorders (45case) is 0.31% and incidence rate is 0.24 (4).
Janus kinase (JAK) 2 is a protein kinase that can add a phosphate group to the signal transducer which will cause the activation of the JAK-STAT route, which ends with expression of several hematopoietic growth factor genes, its mutation cause the development of Myeloproliferative Neoplasms (5). The JAK2-V617F genetic mutation is approximately seventy percent of Myeloproliferative Neoplasm cases. This somatic mutation can cause changing of Valine to phenylalanine at codon 617 (JAK2-V617F) presented in pseudo kinase domain. The repetition of the JAK2-V617F genetic mutation can be presented approximately ninety fife percent in PV, fifty to seventy percent in ET, and forty to fifty percent in PMF. In five percent of JAK2-V617F negative individuals with PV, there is JAK2 exon 12 genetic mutation; but, in ET and PMF there is no such mutation presented (6).
Interleukin 4 (IL-4) is an interleukin cause changing of the naive T helper cells (Th0 cells) to T helper 2 cells. Then T helper 2 cells output more this cytokine using a positive feedback mechanism. Basically the cells that produce IL-4 has not been recognized, but novel researches proposed that may be basophils are those cells. (7) IL-4 has several biological actions, like the activation of B-cell and T-cell proliferation, also the changing of B cells into plasma cells. It is the important organizer in both cellular and humoral immunity. Its cause Bcell class switching to IgE and stimulation of MHC class II production. IL-4 suppresses the outcome of macrophages, T helper 1 cells, IFN-gamma, dendritic cell and IL-12. Excessive amounts of IL-4 can be seen in allergic diseases (8). Many researchers found that Interleukin-4 serum and plasma level is significantly increased in different MPNs types (9) The goal of this study is to estimate IL-4 serum levels in both JAK2-V617F negative and positive mutation in the Iraqi MPNs patients.

Sample collection
Sixty patients were screened by cohort prospective study of having MPN who are patients presented to the Al-Mustansiriyah University / National Center of Hematology, Patients were given their consent verbally, the age of the MPNs patients ranged from thirty to seventy two years old, thirty five males and twenty five females, the methods of diagnosis for MPNs patients including (PV, ET, PMF) depend on; abdominal ultrasound, complete blood picture, blood film, biochemical, molecular (JAK2-V617F mutation), and bone marrow aspirate and biopsy investigations. Depending on the JAK2-V617F mutation, we classified the patients into 3 groups: JAK2-V617F negative (N: 20), JAK2-V617F positive (N: 40) and control group (N: 10). Blood sample (5) ml was obtained from each individual in each group, by venipuncture using disposable syringes for IL-4 serum estimation.

Detection of IL-4 levels by ELISA
ELISA kit (RayBio® -P05112) was applied by using the manual of instructions. In short, the microtiter plate was previously covered with an antibody targeting IL-4 then standards and samples were added to the microtiter plates wells. A biotin-conjugated antibody prepared particularly to IL-4 and avidin conjugated to Horseradish peroxidase (HRP) was poured to every well. After incubation, 3, 3', 5, 5' tetramethyl-benzidine (TMB) substrate solution put in all wells. Specifically, the wells hold interleukine-4 biotin-conjugated antibody avidin going to show an alteration in the dye. The enzyme substrate reaction was stopped by adding (according to the manual), 3 Μ sulphuric acid solution then the dye alteration was measured by a spectrophotometer (ASYS, Australia) with a wavelength of 450 nm ± 2 nm. Finally, IL-4 concentration was estimated by matching the optical density of each sample to the standard curve.

Data Analysis
SPSS were used as descriptive statistics in addition to differences tests using the t test, and the relationships were studied through correlation coefficient.

Demographic Data
The age was ranged (30 -72) years, with 35 men and 25 women, diagnosed as MPN patients including (PV, ET, PMF) as illustrated in Table  1.

Measure the differences between IL-4 serum level in all MPN groups and the control group
A t-test was used in the case of two independent samples to determine whether there was a difference between IL-4 serum level in all MPN samples and the control samples. Table 3 presents the results of the test, where the value of t is 1.315 with significant level (P > 0.05). A clear indication was of no significant differences between IL-4 serum level in all MPN samples and the control samples. Obviously, the dispersion data of IL-4 serum level in JAK2-V617F positive samples was higher than of the control samples. Measure the differences between IL-4 serum level in JAK2-V617F positive group and the control group A t test was used in the case of two independent samples to determine whether there was a difference between IL-4 serum level in JAK2-V617F positive samples and the control samples. Table 5 presents the results of the test where the value of t is 1.420 with significant level (P > 0.05). A clear indication was of no significant differences between IL-4 serum level in JAK2-V617F positive samples and the control samples. Obviously, the dispersion data of IL-4 serum level in JAK2-V617F negative samples was higher than the control samples.

Measure the differences between IL-4 serum level in JAK2-V617F negative group and the control group
A t test was used in the case of two independent samples to determine whether there was a difference between IL-4 serum level in JAK2-V617F negative samples and control samples. Table 7 presents the results of the test where the value of t is 4.391 with significant level (P < 0.05). A clear indication was of significant differences between IL-4 serum level in JAK2-V617F negative samples and control samples.

Measure the differences between IL-4 serum level in JAK2-V617F positive group and IL-4 serum level in JAK2-V617F negative group
A t test was used in the case of two independent samples to determine whether there was a difference between IL-4 serum level in JAK2-V617F positive samples and IL-4 serum level in JAK2-V617F negative samples. Table 8 presents the results of the test where the value of t is 1.411 with significant level (P > 0.05). A clear indication was of no significant differences between IL-4 serum level in JAK2-V617F positive samples and IL-4 serum level in JAK2-V617F negative samples.

Discussion
Onco-inflammation indicates a relationship between tumor and its microenvironment proposing how much important this relation in the beginning and growing of tumors. (10,11) Actually MPNs generally characterized by modified activity of the immune system, expansion of myeloid-derived suppressor cells ,increased monocyte/macrophage compartment, dysfunction of natural killer and T CD4+ cells, and abnormal frequency of regulatory T cells. (12) Increased plasma levels of multiple interleukins and chemokines has been seen in all MPNs in contrast to control groups. That means an inflammatory mechanism might have a role in the physiopathology of MPNs, as chemokines and interleukins act in paracrine, autocrine and endocrine patterns, and may impact the hematopoietic microenvironment which manifested by increased level of IL-4 in all patient groups. (13) Which supported Also by Vaidya, et al. who showed by multiplex beadbased assaying and multivariable analysis ,the increased plasma levels of thirteen interleukins like interlukine-4 was related to the decreased survival in a group of 127 PMF and 65 PV patients. (14) All of the above studies supported our results, which showed obviously an increased mean of Il-4 serum level in All MPNs and both JAK2-V617F Negative and positive patients groups comparing to the control which are reported also in other researches. (15-18) MPNs usually observed with a disorganization of the immune system, and tumor immune evading mechanisms which both collaborated in the evolution and development of the disease. (19) In  (21). Yet lately, interleukin-4 was known to be engaged in tumor invasion and growth. Multiple actions have been characterized, for example the safeguard of cancer cells from programmed death by the stimulation of the anti-apoptotic proteins like survivin which is an apoptosis inhibitor protein (BIRC-5). BIRC-5 stimulated significantly in all three MPNs types. The high serum or plasma level of IL-4 in patients with MPNs may be the main participating element for lowering apoptosis by stimulating several antiapoptotic factors, including survivin. (22,23) Also, IL-4 secreted by M2 macrophage which is well known that it's non-functional against the tumor cells. All these evidences may explain why Il-4 is serum level increased in our study.
In this study a significant differences seen between IL-4 serum level in JAK2-V617F negative samples and control samples which supported by many studies which explained that the prime interleukins are released separately of MPN-linked mutations with manifestation that JAK2-V617F might be delayed incident in MPN progression are stable assuming that long-term activation of myelopoiesis (via inflammation) can forego procuration of genetic mutation in the JAK-2 (CALR and MPL?) genes in the types of MPNs cases. Also some researchers declared that in MPNs, an absence of engagement seen between the JAK2-V617F load and serum or blood estimations of those interleukins. Actually, so likely that it is only a part of those interleukins is guided by JAK2-V617F such as interleukin 4, which plentiful released by other than hematopoietic (nonclonal and nonmutated) cells (24).

Conclusions
The IL-4 serum level is high in MPNs patients, which is one of the immune evading mechanisms of the cancerous acting to imbalance the Th1/Th2 ratio and enhancing the anti-apoptotic activity inside those cells. Increased IL-4 serum levels are independent of the JAK2 status because the JAK2-V617F mutation might be a delayed incident.
Philadelphia-negative myeloproliferative neoplasms as disorders marked by cytokine modulation. Hematology, Transfusion and Cell Therapy.