Clinical significance of interleukin-22 in multiple myeloma George Tsirakis1, Constantina A. Pappa 2, Anna Kolovou 3, Maria Kokonozaki 4, Ioannis Neonakis 4, Michael G. Alexandrakis1,4 1
Hematology Department, University Hospital of Heraklion, Heraklion, Greece, 2Department of Internal Medicine, Venizelion General Hospital of Heraklion, Heraklion, Greece, 3Hematology Department, General Hospital of Chania, Chania, Greece, 4Hematology Laboratory, University Hospital of Heraklion, Heraklion, Greece Objective: Interleukin-22 (IL-22) is a cytokine participating in many aspects of inflammation. Multiple myeloma (MM) is a malignant disease of plasma cells with characteristic immune deregulation. We estimated serum levels of IL-22 in MM patients, both in activity and remission, in order to apprehend its possible participation in MM biology. Methods: We measured serum levels of IL-22 along with beta-2 microglobulin (B2M), paraprotein, and interleukin-1beta (IL-1beta), as well as degree of bone marrow infiltration, in 51 patients with active MM and in 22 of them in remission. Results: We found that IL-22 was higher in active MM patients, compared to both controls and patients in remission, and also in patients in remission compared to controls. Moreover, IL-22 was increasing in parallel with the disease stage and also correlated with B2M, IL1-beta, and degree of infiltration. Discussion: We suggest that the elevated levels of IL-22 in active MM patients, in parallel with disease activity, and in positive correlation with IL-1beta, may represent the inflammatory element of the disease. This increased occurrence of IL-22 may enhance myeloma proliferation and growth, and moreover, may participate in the mechanisms of immune deregulation. Keywords: Angiogenesis, Cytokines, Inflammation, Interleukin-22, Multiple myeloma
Introduction Multiple myeloma (MM) is a plasma cell neoplasm located in the bone marrow (BM). There are major characteristic interactions between myeloma and BM stromal cells. They lead to the production of several factors contributing to dissemination of the disease.1,2 By these means, BM microenvironment alters in parallel with MM progression, favoring the expansion of the clone. There is an inflammatory framework, with the implication of various immune cells and cytokines, leading to significant immune deregulation.3,4 On the other hand, chronic immune stimulation from several autoimmune disorders has been suggested to cause uncontrolled proliferation of plasma cells and therefore MM.5 Although this is rather controversial for the case of rheumatoid arthritis,6–9 it gets more evident for the cases of ulcerative colitis,10 pernicious anemia, and ankylosing
Correspondence to: Michael G. Alexandrakis, Hematology Department, University Hospital of Heraklion, PO Box 1352, Stavrakia, Heraklion 71110, Greece. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1607845414Y.0000000182
spondylitis.9 Therefore, immune deregulation is an important hallmark in myeloma pathophysiology.2 Interleukin-22 (IL-22) is a member of IL-10 cytokine superfamily, which includes potent mediators of cellular inflammatory responses. IL-22 is normally produced by activated Th1-type T, natural killer cells, endothelial, activated dendritic cells, and histiocytes, initiating multiple immune effects.11–14 The transduction of IL-22 signaling is achieved by binding to a heterodimeric receptor complex (IL22R), composed of IL-22R1 and 2, with subsequent activation of intracellular kinases (JAK1, Tyk2, and MAP kinases) and transcription factors, especially STAT3.15,16 By these means, IL-22 has been shown to regulate acute-phase response, activation of the innate immune system, cell migration and differentiation, and gene expression.17–20 IL-22 may be also produced along with IL-17, from splenic lymphoid tissue inducer (LTi)-like cells and T-helper 17 (TH 17) cells, a subset of CD4+ T-cells, in the presence of various pro-inflammatory cytokines, such as IL-6, IL-1beta, IL-21, and IL-23.3,21 These produced
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Clinical significance of IL-22 in MM
cytokines coordinate responses of both adaptive and innate immune systems. They also stimulate inflammatory responses, contributing to various immune diseases.19,20,22 The role of IL-22 in the biology of malignant diseases is not fully understood. For the case of MM, disease progression is characterized by activation of various inflammatory cells and cytokines, capable to stimulate IL-22 production. Moreover, since IL-22 may be involved in the control of cell cycle, it may influence proliferation and subsequently tumor growth.2,23–26 The purpose of the present study is to evaluate serum levels of IL-22 in patients with active MM, before and after effective treatment. In addition, we will try to correlate them with markers of disease activity and burden, such as degree of BM infiltration and serum levels of beta-2 microglobulin (B2M) and paraprotein, as well as with the pro-inflammatory cytokine IL-1beta.
Materials and methods Patients Fifty-one patients with active MM and 18, age- and sex-matched, healthy volunteers were studied. Their characteristics are shown in Table 1. Patients with acute or chronic infection, inflammatory processes, liver or kidney diseases, or incapability to consent were excluded from this study. None of the patients had received any myeloma-related therapy, prior to initial sampling. Twenty-two of the patients responded to anti-MM therapy were also studied (5 in complete response, 17 in very good partial remission; 8 had received PAD (bortezomib, doxorubicin, and dexamethasone), 6 VCD (bortezomib, cyclophosphamide, and dexamethasone), 3 VMP (bortezomib, melphalan, and prednisolone), 3 MP (melphalan and prednisolone), and 2 VAD (vincristine, doxorubicin, and
dexamethasone) regimens) 8–12 weeks after the end of the treatments. The study was performed according to the guidelines of the local Ethics Committee and in accordance with the Declaration of Helsinki (1964). Informed consent for the study was obtained from all subjects.
Methods Serum collected from patients and controls were stored at −70°C. All assays were performed at the end of the study in order to avoid inter-assay variability. Serum levels of IL-22 and IL-1beta were measured with a solid-phase sandwich enzyme-linked immunosorbent assay, using monoclonal human antibody against IL22 and IL-1beta from commercially available test kits (Quantikine ®, R&D Systems Inc., Minneapolis, MN, USA). Patients underwent transiliac BM biopsy, as a routine procedure. The pattern of BM infiltration was highlighted by immunostaining neoplastic plasma cells with a monoclonal antibody to CD38. Monoclonality and percentages of κ/λ neoplastic cells in the BM were assessed by in situ hybridization.
Statistical analyses
Results are expressed as mean ± standard deviation (SD). Statistical comparison between MM patients and healthy controls was made using the Mann–Whitney test. The non-parametric Kruskal–Walis test and the one-way analysis of variance were assessed to test for differences between different stages. The Student–Newman–Keuls test was used for pairwise comparison of the values before and after treatment. Correlations between the parameters measured were calculated with Spearman’s rank correlation coefficient. Values of P < 0.05 were considered to be statistically significant.
Results
35 14 2
Table 2 shows the values of the measured cytokines and B2M in patients with active MM and in healthy population. It can be noted that serum levels of IL22, IL-1beta, and B2M were significantly higher in the group of patients with active myeloma (P < 0.001 for all cases). Table 3 shows the values of the above molecules, as well as percentage of BM by plasma cells and serum levels of paraprotein. It should be noted that the two cases of light-chain MM were excluded in this estimation. It can be seen that in all
15 16 20
Table 2 Mean ± SD serum levels of IL-22, IL-1beta, and B2M in patients with active MM and in healthy controls
Table 1 Characteristics of patients with active myeloma and controls, with mean ± SD values of measured parameters Myeloma patients n = 51
Controls n = 18
25 26 69 (41–87)
9 9 65 (35–78)
Male Female Median age (range) years Paraprotein IgG IgA Light chain ISS stages I II III Skeletal involvement Low score (grades 0–1) High score (grades 2–3) Hemoglobin (g/dl) Serum creatinine (mg/dl) Serum albumin (g/dl)
144
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VOL.
22 29 10.2 ± 3.7 0.97 ± 0.12 3.4 ± 1.4
20
NO.
3
14.9 ± 2.7 0.84 ± 0.21 4.8 ± 1.3
IL-22 (pg/ml) IL-1beta (pg/ml) B2M (mg/l)
MM patients
Controls
P value
75.5 ± 63.3 3.0 ± 1.6 4.5 ± 2.4
8.1 ± 5.7 0.7 ± 0.4 1.3 ± 0.5
Hematology Department, University Hospital of Heraklion, Heraklion, Greece, 2Department of Internal Medicine, Venizelion General Hospital of Heraklion, Heraklion, Greece, 3Hematology Department, General Hospital of Chania, Chania, Greece, 4Hematology Laboratory, University Hospital of Heraklion, Heraklion, Greece Objective: Interleukin-22 (IL-22) is a cytokine participating in many aspects of inflammation. Multiple myeloma (MM) is a malignant disease of plasma cells with characteristic immune deregulation. We estimated serum levels of IL-22 in MM patients, both in activity and remission, in order to apprehend its possible participation in MM biology. Methods: We measured serum levels of IL-22 along with beta-2 microglobulin (B2M), paraprotein, and interleukin-1beta (IL-1beta), as well as degree of bone marrow infiltration, in 51 patients with active MM and in 22 of them in remission. Results: We found that IL-22 was higher in active MM patients, compared to both controls and patients in remission, and also in patients in remission compared to controls. Moreover, IL-22 was increasing in parallel with the disease stage and also correlated with B2M, IL1-beta, and degree of infiltration. Discussion: We suggest that the elevated levels of IL-22 in active MM patients, in parallel with disease activity, and in positive correlation with IL-1beta, may represent the inflammatory element of the disease. This increased occurrence of IL-22 may enhance myeloma proliferation and growth, and moreover, may participate in the mechanisms of immune deregulation. Keywords: Angiogenesis, Cytokines, Inflammation, Interleukin-22, Multiple myeloma
Introduction Multiple myeloma (MM) is a plasma cell neoplasm located in the bone marrow (BM). There are major characteristic interactions between myeloma and BM stromal cells. They lead to the production of several factors contributing to dissemination of the disease.1,2 By these means, BM microenvironment alters in parallel with MM progression, favoring the expansion of the clone. There is an inflammatory framework, with the implication of various immune cells and cytokines, leading to significant immune deregulation.3,4 On the other hand, chronic immune stimulation from several autoimmune disorders has been suggested to cause uncontrolled proliferation of plasma cells and therefore MM.5 Although this is rather controversial for the case of rheumatoid arthritis,6–9 it gets more evident for the cases of ulcerative colitis,10 pernicious anemia, and ankylosing
Correspondence to: Michael G. Alexandrakis, Hematology Department, University Hospital of Heraklion, PO Box 1352, Stavrakia, Heraklion 71110, Greece. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1607845414Y.0000000182
spondylitis.9 Therefore, immune deregulation is an important hallmark in myeloma pathophysiology.2 Interleukin-22 (IL-22) is a member of IL-10 cytokine superfamily, which includes potent mediators of cellular inflammatory responses. IL-22 is normally produced by activated Th1-type T, natural killer cells, endothelial, activated dendritic cells, and histiocytes, initiating multiple immune effects.11–14 The transduction of IL-22 signaling is achieved by binding to a heterodimeric receptor complex (IL22R), composed of IL-22R1 and 2, with subsequent activation of intracellular kinases (JAK1, Tyk2, and MAP kinases) and transcription factors, especially STAT3.15,16 By these means, IL-22 has been shown to regulate acute-phase response, activation of the innate immune system, cell migration and differentiation, and gene expression.17–20 IL-22 may be also produced along with IL-17, from splenic lymphoid tissue inducer (LTi)-like cells and T-helper 17 (TH 17) cells, a subset of CD4+ T-cells, in the presence of various pro-inflammatory cytokines, such as IL-6, IL-1beta, IL-21, and IL-23.3,21 These produced
Hematology
2015
VOL.
20
NO.
3
143
Tsirakis et al.
Clinical significance of IL-22 in MM
cytokines coordinate responses of both adaptive and innate immune systems. They also stimulate inflammatory responses, contributing to various immune diseases.19,20,22 The role of IL-22 in the biology of malignant diseases is not fully understood. For the case of MM, disease progression is characterized by activation of various inflammatory cells and cytokines, capable to stimulate IL-22 production. Moreover, since IL-22 may be involved in the control of cell cycle, it may influence proliferation and subsequently tumor growth.2,23–26 The purpose of the present study is to evaluate serum levels of IL-22 in patients with active MM, before and after effective treatment. In addition, we will try to correlate them with markers of disease activity and burden, such as degree of BM infiltration and serum levels of beta-2 microglobulin (B2M) and paraprotein, as well as with the pro-inflammatory cytokine IL-1beta.
Materials and methods Patients Fifty-one patients with active MM and 18, age- and sex-matched, healthy volunteers were studied. Their characteristics are shown in Table 1. Patients with acute or chronic infection, inflammatory processes, liver or kidney diseases, or incapability to consent were excluded from this study. None of the patients had received any myeloma-related therapy, prior to initial sampling. Twenty-two of the patients responded to anti-MM therapy were also studied (5 in complete response, 17 in very good partial remission; 8 had received PAD (bortezomib, doxorubicin, and dexamethasone), 6 VCD (bortezomib, cyclophosphamide, and dexamethasone), 3 VMP (bortezomib, melphalan, and prednisolone), 3 MP (melphalan and prednisolone), and 2 VAD (vincristine, doxorubicin, and
dexamethasone) regimens) 8–12 weeks after the end of the treatments. The study was performed according to the guidelines of the local Ethics Committee and in accordance with the Declaration of Helsinki (1964). Informed consent for the study was obtained from all subjects.
Methods Serum collected from patients and controls were stored at −70°C. All assays were performed at the end of the study in order to avoid inter-assay variability. Serum levels of IL-22 and IL-1beta were measured with a solid-phase sandwich enzyme-linked immunosorbent assay, using monoclonal human antibody against IL22 and IL-1beta from commercially available test kits (Quantikine ®, R&D Systems Inc., Minneapolis, MN, USA). Patients underwent transiliac BM biopsy, as a routine procedure. The pattern of BM infiltration was highlighted by immunostaining neoplastic plasma cells with a monoclonal antibody to CD38. Monoclonality and percentages of κ/λ neoplastic cells in the BM were assessed by in situ hybridization.
Statistical analyses
Results are expressed as mean ± standard deviation (SD). Statistical comparison between MM patients and healthy controls was made using the Mann–Whitney test. The non-parametric Kruskal–Walis test and the one-way analysis of variance were assessed to test for differences between different stages. The Student–Newman–Keuls test was used for pairwise comparison of the values before and after treatment. Correlations between the parameters measured were calculated with Spearman’s rank correlation coefficient. Values of P < 0.05 were considered to be statistically significant.
Results
35 14 2
Table 2 shows the values of the measured cytokines and B2M in patients with active MM and in healthy population. It can be noted that serum levels of IL22, IL-1beta, and B2M were significantly higher in the group of patients with active myeloma (P < 0.001 for all cases). Table 3 shows the values of the above molecules, as well as percentage of BM by plasma cells and serum levels of paraprotein. It should be noted that the two cases of light-chain MM were excluded in this estimation. It can be seen that in all
15 16 20
Table 2 Mean ± SD serum levels of IL-22, IL-1beta, and B2M in patients with active MM and in healthy controls
Table 1 Characteristics of patients with active myeloma and controls, with mean ± SD values of measured parameters Myeloma patients n = 51
Controls n = 18
25 26 69 (41–87)
9 9 65 (35–78)
Male Female Median age (range) years Paraprotein IgG IgA Light chain ISS stages I II III Skeletal involvement Low score (grades 0–1) High score (grades 2–3) Hemoglobin (g/dl) Serum creatinine (mg/dl) Serum albumin (g/dl)
144
Hematology
2015
VOL.
22 29 10.2 ± 3.7 0.97 ± 0.12 3.4 ± 1.4
20
NO.
3
14.9 ± 2.7 0.84 ± 0.21 4.8 ± 1.3
IL-22 (pg/ml) IL-1beta (pg/ml) B2M (mg/l)
MM patients
Controls
P value
75.5 ± 63.3 3.0 ± 1.6 4.5 ± 2.4
8.1 ± 5.7 0.7 ± 0.4 1.3 ± 0.5
