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Keywords:

  • macrophage inflammatory protein-1 alpha (MIP-1α);
  • multiple myeloma;
  • bone disease;
  • receptor activator of nuclear factor κB

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results and discussion
  5. Correlation of MIP-1α with myeloma bone disease
  6. Correlation of MIP-1α with survival in MM
  7. References

Summary. The role of serum macrophage inflammatory protein-1 alpha (MIP-1α) in bone disease and survival was evaluated in 85 newly diagnosed multiple myeloma (MM) patients. MIP-1α was elevated in MM patients and correlated with the extent of bone disease, bone resorption markers and levels of soluble receptor activator of nuclear factor-κB (RANK) ligand. MIP-1α was also associated with survival; the 3-year probability of survival was 85% and 44% for MIP-1α levels below and above 48 pg/ml respectively (P = 0·021). This suggests that MIP-1α contributes to the pathogenesis of bone disease in MM and possibly in tumour growth, as reflected by its impact on survival.

Macrophage inflammatory protein-1 alpha (MIP-1α) is a low-molecular-weight chemokine that belongs to the RANTES (regulated upon activation, normal T-cell expressed and, presumably secreted) family. It has been characterized recently as a potent osteoclast stimulatory factor in multiple myeloma (MM) (Choi et al, 2000) and acts directly, even on differentiated osteoclasts, in a dose-dependent manner (Han et al, 2001). MIP-1α levels are elevated in bone marrow plasma of patients with active MM and correlate with the presence of lytic lesions (Choi et al, 2000; Abe et al, 2002). The production of MIP-1α by plasma cells was documented by measuring the supernatant of cultured bone marrow cells of MM patients by enzyme-linked immunosorbent assay (ELISA) or by the presence of MIP-1α m-RNA in plasma cells (Abe et al, 2002; Uneda et al, 2003). Apart from its effect on osteoclast activation, it stimulates proliferation, migration and survival of plasma cells (Lentzsch et al, 2003). Blocking MIP-1α activity by either neutralizing antibodies or antisense oligonucleotides resulted in a reduction in bone disease as well as tumour burden in MM animal models (Choi et al, 2001).

In the present study, we have evaluated the role of serum MIP-1α in bone disease and survival in MM patients.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results and discussion
  5. Correlation of MIP-1α with myeloma bone disease
  6. Correlation of MIP-1α with survival in MM
  7. References

Eighty-five newly diagnosed, previously untreated, MM patients (41M/44F) with a median age of 68 years (range 36–78 years) were studied. Patients were diagnosed between 1992 and 2000 and the serum samples were evaluated retrospectively. MIP-1α was measured by ELISA (Amersham Pharmacia Biotech, Little Chalfont, UK). Soluble receptor activator of nuclear factor-κB ligand (sRANKL), osteoprotegerin (OPG), markers of bone resorption [N-terminal telopeptide of type-I collagen (NTX), tartrate-resistant acid phosphatase isoform-5b (TRACP-5b)], markers of bone formation [osteocalcin (OC), bone alkaline phosphatase (bALP)] and markers of disease activity [β2-microglobulin, C-reactive protein (CRP), interleukin 6 (IL-6)] were also evaluated. The above parameters were also measured in 12 age- and sex-matched controls. The extent of bone disease was assessed radiographically, and grading was performed as follows: group A included 10 patients who had no lytic lesions and/or osteoporosis, group B included 27 patients who had one to three lytic lesions, whereas group C included 48 patients with more than three lesions and/or a bone fracture.

Survival probabilities were calculated by the Kaplan–Meier method. Associations between bone disease and biochemical markers were examined by the Kruskal–Wallis test, while the Spearman rank correlation test was used to examine relationships between various parameters.

Correlation of MIP-1α with myeloma bone disease

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results and discussion
  5. Correlation of MIP-1α with myeloma bone disease
  6. Correlation of MIP-1α with survival in MM
  7. References

MIP-1α serum levels were elevated in MM patients, but there was no significant difference between patients and controls (mean values: 32·0 pg/ml versus 22·7 pg/ml, P = 0·15). However, the levels of MIP-1α correlated with the extent of bone disease as graded above (P < 0·0001; Fig 1A). Serum levels of MIP-1α also correlated with NTX (r = 0·594, P < 0·0001; Fig 1B), TRACP-5b (r = 0·376, P < 0·0001; Fig 1C), sRANKL (r = 0·382, P < 0·0001; Fig 1D) and β2-microglobulin (r = 0·401, P < 0·0001; Fig 1E). MIP-1α did not correlate with the other markers of bone remodelling, and haemoglobin, creatinine, albumin, paraprotein, CRP or IL-6.

image

Figure 1. (A) Correlation of MIP-1α levels with the extent of bone disease as assessed by radiographic evaluation (P < 0·0001). Patients in groups A and B had lower median values of MIP-1α than patients in group C (11·5 pg/ml versus 20·5 pg/ml, P < 0·02). (B–E) Correlation of MIP-1α levels with: (B) NTX levels (r = 0·594, P < 0·0001); (C) TRACP-5b levels (r = 0·376, P < 0·0001); (D) sRANKL levels (r = 0·382, P < 0·0001); (E)  β 2-microglobulin levels (BETA2-MG, r = 0·401, P < 0·0001).

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TRACP-5b is an enzyme that is produced only by activated osteoclasts, while NTX is a resorption marker that not only reflects the extent of bone disease but also predicts bone disease progression during chemotherapy (Abildgaard et al, 2003; Terpos et al, 2003). These correlations confirm, for the first time in humans, the role of MIP-1α in osteoclast activation and the subsequent increase in bone resorption that leads to the development of lytic lesions in MM.

In our study, MIP-1α was detected in blood serum, and nine patients had values > 48 pg/ml (the upper limit for the normal controls set by the manufacturer). All nine of these patients had stage III disease and belonged to group C in terms of bone disease. In previous studies, levels of MIP-1α were elevated in the bone marrow plasma of the majority (62%) of patients with active disease, but it was not detected in the peripheral blood (Choi et al, 2000). Uneda et al (2003) have shown that 58% of MM patients expressed MIP-1α mRNA and suggested that this expression may contribute to the development of bone lesions, irrespective of the serum levels of the protein. However, it is not clear how the mere presence of mRNA could lead to osteoclast activation without the concomitant increase in the respective protein. The strong correlation of MIP-1α with severe bone disease (group C) in our cohort of patients could support the hypothesis that, although bone destruction is a local process, high levels of MIP-1α can be achieved and detected in the serum if the bone disease is sufficiently severe.

Han et al (2001) reported that MIP-1α activity was independent of RANKL, although it enhanced the effect of RANKL on osteoclast activation, and high concentrations of the soluble form of the RANK receptor (RANK-Fc) inhibited osteoclast formation induced by MIP-1α. In contrast, Abe et al (2002) reported that MIP-1α induced expression of RANKL by mouse marrow stromal cells. We have shown that MIP-1α levels correlated with those of sRANKL. This in vivo finding indirectly supports the notion that MIP-1α enhances osteoclast activation and, subsequently, bone resorption through the RANKL pathway, which is believed to be the dominant, final mediator of osteoclast activity in myeloma bone disease.

Correlation of MIP-1α with survival in MM

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results and discussion
  5. Correlation of MIP-1α with myeloma bone disease
  6. Correlation of MIP-1α with survival in MM
  7. References

After diagnosis, 59 patients received conventional chemotherapy, while 28 patients underwent autologous stem cell transplantation. The median survival of our group of patients was 4·7 years. Elevated levels of MIP-1α were associated with survival in the univariate analysis. Patients with MIP-1α serum levels below 48 pg/ml had a probability of survival of 85% at 3 years, whereas patients with MIP-1α levels > 48 pg/ml had a 44% probability of survival at 3 years (P = 0·021) (Fig 2). The multivariate analysis revealed that only the ratio of sRANKL/OPG, β2-microglobulin and CRP were independent prognostic factors, possibly because of the strong correlation of MIP-1α with sRANKL and β2-microglobulin as well as the low number of patients (nine) with abnormal MIP-1α values (> 48 pg/ml).

image

Figure 2. Probability of survival of MM patients for MIP-1α.

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The effect of MIP-1α on survival can be explained by its impact on plasma cell accumulation and proliferation (Lentzsch et al, 2003). Furthermore, treatment of a MM mouse model with a monoclonal rat anti-mouse MIP-1α antibody resulted not only in a significant reduction in bone lesions but also in a significant reduction in serum paraprotein (Oyajobi & Mundy, 2003). These results, together with our observation that MIP-1α levels correlate with survival, suggest that the MIP-1α pathway may be a target for novel antimyeloma treatment regimens.

In conclusion, our data implicate MIP-1α in the pathogenesis of myeloma bone disease and possibly in tumour growth as reflected by its impact on survival.

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results and discussion
  5. Correlation of MIP-1α with myeloma bone disease
  6. Correlation of MIP-1α with survival in MM
  7. References
  • Abe, M., Hiura, K., Wilde, J., Moriyama, K., Hashimoto, T., Ozaki, S., Wakatsuki, S., Kosaka, M., Kido, S., Inoue, D. & Matsumoto, T. (2002) Role for macrophage inflammatory protein (MIP)-1alpha and MIP-1beta in the development of osteolytic lesions in multiple myeloma. Blood, 100, 21952202.
  • Abildgaard, N., Brixen, K., Kristensen, J.E., Eriksen, E.F., Nielsen, J.L. & Heickendorff, L. (2003) Comparison of five biochemical markers of bone resorption in multiple myeloma: elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for early progression of the bone disease during standard chemotherapy. British Journal of Haematology, 120, 235242.
  • Choi, S.J., Cruz, J.C., Craig, F., Chung, H., Devlin, R.D., Roodman, G.D. & Alsina, M. (2000) Macrophage inflammatory protein 1-alpha is a potential osteoclast stimulatory factor in multiple myeloma. Blood, 96, 671675.
  • Choi, S.J., Oba, Y., Gazitt, Y., Alsina, M., Cruz, J., Anderson, J. & Roodman, G.D. (2001) Antisense inhibition of macrophage inflammatory protein 1-alpha blocks bone destruction in a model of myeloma bone disease. Journal of Clinical Investigation, 108, 18331841.
  • Han, J.H., Choi, S.J., Kurihara, N., Koide, M., Oba, Y. & Roodman, G.D. (2001) Macrophage inflammatory protein-1alpha is an osteoclastogenic factor in myeloma that is independent of receptor activator of nuclear factor kappa B ligand. Blood, 97, 33493353.
  • Lentzsch, S., Gries, M., Janz, M., Bargou, R., Dorken, B. & Mapara, M.Y. (2003) Macrophage inflammatory protein-1alpha (MIP-1{alpha}) triggers migration and signaling cascades mediating survival and proliferation in multiple myeloma (MM) cells. Blood, 101, 35683573.
  • Oyajobi, B.O. & Mundy, G.R. (2003) Receptor activator of NF-kappaB ligand, macrophage inflammatory protein-1alpha, and the proteasome. Cancer, 97, 813817.
  • Terpos, E., De La Fuente, J., Szydlo, R., Hatjiharissi, E., Viniou, N., Meletis, J., Yataganas, X., Goldman, J.M. & Rahemtulla, A. (2003) Tartrate-resistant acid phosphatase isoform 5b: a novel serum marker for monitoring bone disease in multiple myeloma. International Journal of Cancer, 106, 455457.
  • Uneda, S., Hata, H., Matsuno, F., Harada, N., Mitsuya, Y., Kawano, F. & Mitsuya, H. (2003) Macrophage inflammatory protein-1 alpha is produced by human multiple myeloma (MM) cells and its expression correlates with bone lesions in patients with MM. British Journal of Haematology, 120, 5355.