Farrugia et al. (1) in the September 1, 2003 issue of Cancer Research have arisen an interesting and controversial question about the direct expression or production of the critical osteoclastogenetic receptor activator of nuclear factor κB ligand (RANKL) by human myeloma cells. Farrugia et al. (1) have shown that sorted CD38+++ subpopulation expressed RANKL protein by flow cytometry and RANKL mRNA, as well as its receptor, receptor activator of nuclear factor κB, mRNA by reverse transcription-PCR. Moreover, in a coculture system with peripheral blood mononuclear cell they show that sorted CD38+++ cells induced osteoclast formation that is abolished by osteoprotegerin (OPG) treatment. On the basis of their data the authors conclude that myeloma cells directly produce RANKL and that RANKL is directly involved in the osteoclast formation induced by myeloma cells. These data are in contrast with the evidence reported by our group (2, 3) and other authors (4, 5, 6). In particular we found that purified CD138-positive myeloma cells of 26 patients in different stage of disease were negative for RANKL mRNA expression using a specific primer pair that recognizes both trans-membrane and soluble RANKL isoform (2). Moreover we found that several human myeloma cell lines (RPMI-8226, U266, OPM-2, LP-1, XG-1, XG-6, MDN, and JJN3) did not express RANKL and receptor activator of nuclear factor κB mRNA or produced soluble RANKL (2, 3). Similarly, Pearse et al. (4) tested ARP-1, U266, RPMI-8226, H929, and EBV-positive ARH-77 cell lines, and failed to find RANKL mRNA or protein. Moreover, RANKL immunostaining, performed by different authors on bone marrow biopsies of multiple myeloma (MM) patients has shown that myeloma cells are negative for RANKL expression (1, 4, 5, 6). In line with these observations, Shaughnessy et al. (6), using the microarray technology, confirmed recently that RANKL has not been detected in purified CD138-positive MM cells of either 83 osteolytic or 87 nonosteolytic MM patients, as well as in normal bone marrow plasma cells. All of these experimental evidences demonstrate that human myeloma cells do not express or directly produce the critical osteoclastogenetic factor RANKL in contrast with the observations of Farrugia et al(1). Why is there this discrepancy? Could it be due to the different type of isolation of plasma cells?

We have some observations and questions about the methodologies and the conclusions presented by the authors.

First they have sorted and tested CD38+++ cells instead of CD138-positive cells. CD138 is widely accepted as specific marker for myeloma cells and it is usually used to isolate MM cells (7). Sorting plasma cells by CD38 antigen expression alone the authors cannot exclude the presence of other contaminating cells that are known to express RANKL such as, in particular, activated T lymphocytes (2, 8). Did the authors test RANKL mRNA and protein on CD138-positive cells and on human myeloma cell lines that are a model of high purified MM cells? Moreover, the monoclonal antibody used to detect RANKL by flow cytometry (MAB626; R&D), the same used by Heider et al. (9), is not validated for flow cytometry application. So, the authors should prove the specificity of this mAb.

When the authors investigate the RANKL mRNA expression in sorted cells on the basis of CD38 expression they show that both CD38+ and CD38+++ express RANKL. Thus, how could they explain that CD38+ cells do not induce osteoclast formation in their system in contrast to CD38+++ cells? Anyway, because both CD38+, which are not myeloma cells, and CD38+++ cells express RANKL, we could suppose that the direct RANKL production by myeloma is not a critical determinant in myeloma-induced osteoclast formation.

In the article the authors say that they have used a more sensitive PCR technology in comparison with our method (2, 3). But, if they had to increase the sensitivity of PCR to obtain RANKL mRNA signal in plasma cells, they support the hypothesis that RANKL expression in CD38+++ cells is very low. Moreover, using a more sensitive method they could have a high risk to amplify cDNA derived from contaminating cells considering that they do not use CD138 antibody to isolate plasma cells.

The last point is about the osteoclastogenetic system. Using all of the peripheral blood mononuclear cells to generate osteoclasts, instead of purified CD14+ monocytes (10), the authors are not able to demonstrate that the direct RANKL production by CD38+++ is critical in osteoclast formation, because T lymphocytes are present in their system and it has been demonstrated that myeloma cells up-regulate RANKL in T lymphocytes.

Moreover, the osteoclastogenetic effect observed by the authors using the CD38+++ cells is little as compared with what is observed by myeloma cells in the presence of stromal cells, suggesting that stromal cells are critical in the osteoclastogenetic effect of myeloma cells (1, 11).

Several evidence have demonstrated that RANKL/OPG expression and ratio in stromal/osteoblastic cells is the critical point in the regulation of osteoclast formation both in physiological and pathophysiological conditions (12). Moreover a role of T lymphocytes has been postulated recently in the regulation of bone resorption through RANKL expression (8). In line with these observations, different groups have shown that myeloma cells induce RANKL and inhibit its soluble antagonist OPG in bone marrow stromal/osteoblastic and T cells both in a coculture system and in vivo in MM patients. Moreover, immunohistochemistry, performed on bone marrow biopsies by several authors, has also confirmed the presence of an imbalance in RANKL/OPG expression in the bone marrow environment. All of these evidence strongly suggest that RANKL/OPG system is mainly involved in the activation of osteoclastic cells by myeloma cells indirectly through the bone marrow environment.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Nicola Giuliani, Chair of Hematology and BMT Unit, University of Parma, via Gramsci 14, 43100 Parma, Italy. Phone: 00390 521290787; Fax: 00390 521292765; Email: [email protected] or [email protected]

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