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|Year : 2014 | Volume
| Issue : 2 | Page : 141-144
IL-21 and other serum proinflammatory cytokine levels in patients with multiple myeloma at diagnosis
O Mehtap1, EB Atesoglu1, P Tarkun1, A Hacihanefioglu1, I Dolasik1, MM Musul2
1 Department of Haematology, Kocaeli University Medical Faculty, Kocaeli, Turkey
2 Department of Biochemistry, Kocaeli University Medical Faculty, Kocaeli, Turkey
|Date of Submission||07-Mar-2013|
|Date of Decision||09-Nov-2013|
|Date of Acceptance||03-Jan-2014|
|Date of Web Publication||13-May-2014|
Dr. O Mehtap
Department of Haematology, Kocaeli University Medical Faculty, Kocaeli
Source of Support: None, Conflict of Interest: None
Background: IL-6, IL1-β, TNF-α and IL-21 have been identified in the growth, progression and dissemination of multiple myeloma. To dte, there is no published data about serum levels of IL-21 in patients with multiple myeloma. In the present study we have investigated circulating levels of cytokines, such as IL-6, IL-1β, TNF-α, IL-21 and the association of these levels with the disease stage in newly diagnosed multiple myeloma patients. Materials and Methods: Twenty healthy controls and 44 newly diagnosed multiple myeloma patients were evaluated. Patients were classified according to Durie-Salmon criteria, international staging system (ISS) and bone disease. Quantification of cytokine levels in serum were performed by using ELISA. Results: The levels of cytokines in patients' serum are found elevated than healthy controls. However, only the serum levels of IL-1β and TNF-α were found statistically significant. TNF-α levels of patients with ISS stage 3 were significantly higher than patients with ISS stage 1 and 2 (P 0.000). IL-1β was significantly elevated in advanced stage patients (stage II-III) (P 0.040). There was no correlation between IL-1β, TNF-α, IL-21 levels and bone lesions. IL-6 levels were significantly elevated who have at least three visible lytic bone lesions and/or bone fracture in comparison to patients who have one or two visible or no visible lytic bone lesions (P 0.048). Conclusion: It appears that there is no association of serum IL-21 level with multiple myeloma in contrast to the other cytokines such as IL-6, IL-1β, TNF-α.
Keywords: IL-1β, IL-6, IL-21, Multiple myeloma, TNF-α
|How to cite this article:|
Mehtap O, Atesoglu E B, Tarkun P, Hacihanefioglu A, Dolasik I, Musul M M. IL-21 and other serum proinflammatory cytokine levels in patients with multiple myeloma at diagnosis. J Postgrad Med 2014;60:141-4
|How to cite this URL:|
Mehtap O, Atesoglu E B, Tarkun P, Hacihanefioglu A, Dolasik I, Musul M M. IL-21 and other serum proinflammatory cytokine levels in patients with multiple myeloma at diagnosis. J Postgrad Med [serial online] 2014 [cited 2020 Apr 6];60:141-4. Available from: http://www.jpgmonline.com/text.asp?2014/60/2/141/132319
| :: Introduction|| |
Multiple myeloma is a fatal disease, characterized by the neoplastic proliferation of immunoglobulin-producing plasma cells in bone marrow. A cytokine network has been identified in the growth, progression and dissemination of multiple myeloma.  Initial reports about IL-6 have revealed that there is an enormous effect of IL-6 on the survival and proliferation of multiple myeloma cells. , Like the critical role of IL-6, IL-1β and TNF-α are both known to be potent osteoclast activators in myeloma pathogenesis. , In addition to this osteolytic activity, it has been shown that IL-1β and TNF-α both augment the production of IL-6 in human myeloma and bone marrow stromal cells.  The effect of TNF-α on proliferation and differentiation of myeloma cells are suggested by other studies. ,
Interleukin-21 (IL-21), mainly sourced from activated T lymphocytes, was first described a decade ago. This cytokine has a significant sequence homology to IL-2, IL-4 and, in particular, IL-15. It was suggested that IL-21 has a proliferative effect and could modulate the function of T, B and NK cells. , After these investigations, Brenne et al. demonstrated IL-21, as a growth and survival factor for myeloma cells.  Later on, the effect of IL-21 on B cells was more clarified and it was shown that IL-21 induces the differentiation of B cells into plasma cells through up-regulation of BLIMP1.  Since then, many studies with distinctive results have been published about serum levels of IL-6, IL-1β and TNF-α in multiple myeloma patients. To date, there is no published data about serum levels of IL-21 in patients with multiple myeloma. In the present study we have investigated circulating levels of cytokines, such as IL-6, IL-1β, TNF-α, IL-21 and the association of these levels with the disease stage in newly diagnosed patients of multiple myeloma.
| :: Materials and Methods|| |
The study was conducted according to good clinical and laboratory practice rules and the principles of the Declaration of Helsinki, and approved by the local ethics committee, and also written informed consents were obtained from all of the participants. The study included 20 (seven female and 13 male) healthy controls who were admitted in regular checkup clinic and 44 (21 female and 23 male) newly diagnosed multiple myeloma patients who were identified in the Haematology Department between February 2010 to February 2011. Individuals who did not have any disease and did not receive any medicals were considered as healthy controls. The groups were age matched and blood samples were collected from all groups. Patients were classified into three stages according to Durie-Salmon criteria and international staging system (ISS). And the patients were also sub-grouped according to their bone disease. All patients' lytic bone lesions and/or pathological fractures were determined by skeletal X-ray survey. The patients, who have active infection signs and symptoms, were not included in the study in case active infection could affect the level of cytokines. Quantification of IL-6 was performed using IL-6-EASIA-CE (DIAsourse ImmunoAssays Belgium). In the manual of this ELISA kit, reference intervals were given as 0-50 pg/ml. IL-1β-EASIA (DIAsourse ImmunoAssays Belgium) kit, which was used for determination of IL-1β levels with reference intervals of 0-17 pg/ml. TNF-α levels in serum of the cases were determined with TNF-α-EASIA (DIAsourse ImmunoAssays Belgium) kit, with reference intervals ranging between 4.6 and 12.4 pg/mL. For measurement of IL-21 levels Human IL-21 Platinum ELISA (eBioscience) kit was used and in the manufactures' guidelines the sensitivity of this test was determined to be 20 pg/mL and it is denoted that in sera of 39 out of 40 healthy donors, IL-21 was at an undetectable level and in only one sample IL21 was found to be 227.6 pg/mL.
All analyses were performed using Statistical Package for Social Sciences statistical package (SPSS, version 15.0 for Windows XP, Chicago, IL, USA).Median and ranges were calculated for each parameter. According to the distribution of the results, nonparametric and parametric tests were done for measuring significance. In this context ANOVA test, T test, Mann Whitney U test and Kruskall-Wallis test was performed. When ANOVA test was found significant, post hoc test was done to measure the event that was performed. P values <0.05 were considered statistically significant.
| :: Results|| |
The patients' characteristics are described in [Table 1]. Results of serum cytokines measurements are given in [Table 2]. As it was shown in [Table 2], the levels of cytokines in patients' serum were found to be higher in comparison to healthy controls. However, only the serum levels of IL-1β and TNF-α were found to be significantly different.
The P values, in the consideration of stage, ISS and bone disease are summarized in [Table 3]. The ANOVA test revealed that there is a correlation between TNF-α and ISS stages (P 0.00). After evaluation with post hoc test, TNF-α level of patients with ISS stage 3 were significantly higher than patients with ISS stage 1 and 2 (P 0.00 for ISS stage 3 vs stage 1 and P 0.01 for ISS stage 3 vs stage 2, not shown in table).
The comparisons of early (stage I) and advanced (stage II-III) disease P values are also presented in [Table 3]. Mann Whitney test and T test was performed to measure the significance between cytokine levels and early (stage I) or advanced stages (stage II-III). IL-1β levels were significantly elevated in advanced stage patients. There was no correlation between IL-1 β, TNF-α, IL-21 levels and bone lesions. However, IL-6 levels were significantly elevated who have at least three visible lytic bone lesions and/or bone fracture in comparison to patients who have 1-2 visible or no visible lytic bone lesions.
| :: Discussion|| |
IL-6 can be produced by different cell types and overproduction of IL-6 has been demonstrated in malignancies, chronic and acute infections. ,,,, In a study conducted by Bataille et al., IL-6 levels were elevated in 37% of multiple myeloma patients at diagnosis which was significant. Increased serum IL-6 levels were found in patients with high tumor mass (stage III) or in those with high serum, β2 microglobulin levels (>6 mg/L) suggesting an association with the proliferation of malignant plasma cells.  In another study, correlation was found between the stage of the disease and the level of IL-6 at the time of diagnosis determining IL-6 as a prognostic factor in multiple myeloma.  In accordance with these studies, Merico et al. have detected low levels of serum IL-6 in four of 30 cases, usually in advanced stages of the disease in their study. They suggested that IL-6 is produced locally within the bone marrow and occasionally may leak into the circulation in multiple myeloma.  In contrast to these studies, the serum levels of IL-6 which was measured by ELISA in normal volunteers, patients with multiple myeloma and benign monoclonal gammopathies exhibit statistically insignificant differences.  Moreover, Ballester et al. determined that high levels of IL-6 are associated with low tumor burden and low growth fraction in patients with multiple myeloma.  In a review article, it has been emphasized that these differences may reflect the different methods that have been used to evaluate IL-6 activity, particularly between bioassay and levels measured by ELISA; also it has been emphasized that the samples negative by ELISA may prove positive by bioassay in the same paper.  In our study, we found increased serum IL-6 levels in patients with multiple myeloma than normal volunteers; however, this was not statistically significant. As mentioned above, this result could be related to the method for assessing IL-6 levels. Another point of view is to consider that we did not include the myeloma patients with active infection signs and symptoms to the study. Patient with multiple myeloma can present with active infection at initial diagnosis and as discussed above, the serum IL-6 levels could be affected by active infection which could increase serum IL-6 levels.  Studies which have found increased serum IL-6 levels significant, did not inform about patients' active infection status and whether they have included patients with active infection in their study or not. ,, The method we used for determination of IL-6, the infection status or the limited number of patients enrolled to this study could be the reason of our insignificant results. On the other hand, we determined that the serum levels of IL-6 were significantly higher, in patients who have at least three visible lytic bone lesions and/or bone fracture than the patients who have 1-2 visible or no visible lytic bone lesions; which is probably the effect of IL-1β of TNF-α on IL-6 secretion in advanced multiple myeloma patients. Furthermore, it is known that myeloma cell-stromal cell interaction induces RANKL and IL-6 production in bone marrow microenvironment. Induced IL-6 production contributes to osteoclast activation and bone resorption.  This mechanism may explain the correlation between IL-6 and bone lesions in our study.
Both IL-1β and TNF-α, are potent bone-resorbing proinflammatory cytokines that may contribute to the development of osteolytic bone lesions in patients with multiple myeloma. , In a study Carter et al. determined that production of IL-6 by human myeloma cells could be increased by IL-1β and TNF-α. In addition to this finding, they also showed that DNA synthesis occurring in response to culture supernatants of human myeloma cells exposed to IL-1β or TNF-α were significantly greater than to media conditioned by unstimulated myeloma cells.  These findings attracted the investigators' attention to search the serum levels of IL-1β and TNF-α in patients with multiple myeloma. Different results were found about serum levels of IL-1β and TNF-α in different studies of multiple myeloma patients. Merico et al. emphasized that TNF-α was above the threshold detection level (10 pg/ml) in only one of 30 multiple myeloma cases and likewise undetectable in 24 normal donors. In addition, they also showed that IL-1β was above the threshold detection level (20 pg/ml) in only one of 30 multiple myeloma cases and could not be detected in 24 normal donors.  Contrary to these results, in a study Kuku et al. determined significant higher serum IL-1β and TNF-α level in patients with multiple myeloma than control groups.  Similar to this study, our results showed significant higher IL-1β and TNF-α levels than control groups. Apart from this finding, we also demonstrated that the patients with advanced disease have significant high TNF-α level according to the ISS. In the consideration of Durie-Salmon staging system, we found that IL-1β levels are higher in advanced stage multiple myeloma patients than stage I patients. These results could be explained by the effect of tumor burden on IL-1β and TNF-α.
IL-21 is the other cytokine, which affects the myeloma cells survival. Brenne et al. demonstrated IL-21 induced proliferation of myeloma cells in their study. They showed this proliferation was dose dependent and moreover they indicated that IL-21 could replace IL-6 as a long-term growth factor in myeloma cell line. Beside this proliferation effect, the authors emphasized two other function of IL-21; first, IL-21 was an anti-apoptotic factor for human cell lines and second, IL-21 and TNF-α had synergistic effects on myeloma cell proliferation.  The studies, about IL-21, conducted by Brenne et al. and Ozaki et al. were performed on multiple myeloma cell lines in vitro at laboratory. , Until now no study has been published about serum levels of IL-21 in patients with multiple myeloma. In our study, we demonstrated higher serum IL-21 levels in myeloma cases than healthy control group however it was not statistically significant. In addition, we did not find any association with serum levels of IL-21 and disease stage (neither Durie-Salmon nor ISS). These results could be explained by a number of questions; 1) Does IL-21 exist in bone marrow much more than in peripheral blood? We do not know about IL-21 levels in bone marrow which affects plasma cells locally. 2) Does minimal amount of IL-21 affect malignant plasma cells? Even, physiologic levels of IL-21 could be adequate for affecting malignant plasma cells. 3) Does blood IL-21 has oscillation which could change time to time like IL-6? It was established that serum IL-6 levels show daily diurnal variations in some patients. 
In conclusion, the serum cytokines, especially IL-6, in myeloma patients seem to be controversial. Because of the reasons mentioned above, serum levels of cytokines could change for each study. As Doctor Lauta emphasized, a single measurement of serum IL-6 should be interpreted with caution and confirmed by multiple determinations over a period of several days.  Currently, no study about IL-21 level in multiple myeloma patients exists in literature. According to our study, it seems that there is no association of serum IL-21 level with multiple myeloma in contrast to the other cytokines such as IL-6, IL-1β, TNF-α. And also, it seems that measurement of serum IL-21 is not beneficial in clinical practice. Our study has some limitations such as decreased number of patients and difficulties for reaching a conclusion by serum cytokine measurements as there are many factors affecting the levels of cytokines in patients. However, the importance of IL-21, in multiple myeloma could be highlighted by new comprehensive investigations in the future.
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[Table 1], [Table 2], [Table 3]
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