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Blood, 15 March 2002, Vol. 99, No. 6, pp. 2277-2278
CORRESPONDENCE
To the editor:
Role of surface IgM and IgD on survival of the cells from
B-cell chronic lymphocytic leukemia
We read with great interest the paper by Bernal et
al1 on the ability of anti-human IgM antibodies to prevent
spontaneous apoptosis of B-cell chronic lymphocytic leukemia
(B-CLL) cells in vitro. These findings suggest that receptor engagement
by certain antigens, perhaps autoantigens that are constantly available
in vivo, may contribute to the survival of the neoplastic cells. The data, although interesting, are in contrast with previous findings
of other groups including our own that showed that exposure to anti-µ
antibodies causes rapid apoptosis of certain B-CLL
cells.2-4 More recently we have also demonstrated that when an anti-Ig- chain antibody is used, prolonged cell survival instead of apoptosis is observed.5 From these findings it
appears that the specificity of the antibodies to surface Ig plays a
crucial role in the outcome of the experiments. Unfortunately, in the experiments by Bernal et al1 very little attention is paid to the specificity of the reagent used. If the anti-Ig reagent (a
polyclonal goat anti-IgM antibody) that they used also reacted with light chains then the cells could have been stimulated via surface
IgM and IgD. In these conditions, the prevailing physiological pathway
would be survival or apoptosis depending on which of the 2 signals is
more potent. Differences of about 15% in the Annexin-V staining are barely
significant with the presently used methods, according to the experience accumulated by several groups including our own. Thus, 3 B-CLL groups can be distinguished in the work of Bernal et al1(Tab
1): those that do not respond at all to anti-IgM treatment
(cases 31, 86, 89); those that barely respond (cases 47, 69, 72, 96, 104, 106, 108, 111, 114, 121); and those who definitely respond (cases
58, 78). The conclusions of the authors, as mentioned in the title of
their article, are based primarily upon the latter 2 cases. Bernal et al state that for our previous work we selected for cases
expressing CD38. These cases might represent a special subset
of B-CLL. CD38+ B-CLL cells represent 30% to 60%
of all the B-CLL (depending upon the different cohorts
studied).6 Even before CD38 became a fashionable marker,
it was already known that the cells from only about half of the B-CLL
cases responded to anti-Ig stimulation in vitro as assessed in
Ca++ mobilization or tyrosine kinase phosphorylation
assays.7,8 We just pointed out that a correlation existed
between CD38 expression and viability of the signal transduction
pathway. Based upon these considerations the question arises of how
many of the cases utilized by Bernal et al had a functional
IgM/IgD-dependent signal transduction pathway initiated by surface Ig
cross-linking. This is not trivial, considering the general
implications of the paper brought about by their title. A final comment concerns the observation reported in the paper that
certain genes potentially related to antiapoptotic activity are
up-regulated following exposure of B-CLL cells to anti-IgM antibodies.
This up-regulation (which is not very dramatic as apparent from the
figures shown by Bernal et al) should not inevitably be taken as
evidence for the fact that anti-IgM stimulation causes activation of an
antiapoptotic program. In studies that we have not published,
we have compared the capacity of anti-µ chain or anti-
chain stimulation to promote the synthesis of a variety of molecules
(M.M. and S.Z., unpublished data, May 2001). There is virtually
comparable up-regulation of the same molecules studied by Bernal et al
following exposure to anti- or anti-µ reagents. However, the
2 types of stimuli had a remarkably different effect on caspase
activation and consequently on apoptosis. In conclusion, the interesting hypothesis proposed by Bernal et al does
not appear to be sustained by the available experimental data. It is
possible that B-CLL cells are exposed to a variety of apoptotic and
antiapoptotic signals in vivo and that the survival of a large fraction
of them depends upon the fact that the antiapoptotic signals are prevailing.
Simona Zupo, Giovanna Cutrona, Massimo Mangiola, and Manlio Ferrarini
Correspondence: Simona Zupo, Istituto Nazionale per la Ricerca
sul Cancro, Servizio di Immunologia Clinica, Genoa, Italy
References
1.
Bernal A, Pastore RD, Asgary Z, et al.
Survival of leukemic B cells promoted by engagement of the antigen receptor.
Blood.
2001;98:3050-3057[Abstract/Free Full Text].
2.
McConkey DJ, Aguillar-Santelises M, Hartzell P, et al.
Induction of DNA fragmentation in chronic B-lymphocytic leukemia cells.
J Immunol.
1991;146:1072-1076[Abstract].
3.
Osorio LM, Santiago A, Aguillar-Santelises M, Mellstedt H, Jondal M.
CD6 ligation modulates the bcl2/bax ratio and protects chronic lymphocytic leukemia B cells from apoptosis induced by anti-IgM.
Blood.
1997;89:2833-2841[Abstract/Free Full Text].
4.
Zupo S, Isnardi L, Megna M, et al.
CD38 expression distinguishes two groups of B-cell chronic lymphocytic leukemias with different responses to anti IgM antibodies and propensity to apoptosis.
Blood.
1996;87:2615-2625[Free Full Text].
5.
Zupo S, Massara R, Dono M, et al.
Apoptosis or plasma cell differentiation of CD38 positive B-chronic lymphocytic leukemia cells induced by cross-linking of surface IgM or IgD.
Blood.
2000;95:1199-1206[Abstract/Free Full Text].
6.
Chiorazzi N, Ferrarini M.
Immunoglobulin variable region gene characteristics and surface membrane phenotype define B-CLL subgroups with distinct clinical courses. In:
Cheson B, ed.
Chronic lymphocytic leukemia. 2nd ed. New York, NY: Marcel Dekker; 2001:81-109.
7.
Hivroz C, Geny B, Brouet JC, Grillot-Courvalin C.
Altered signal transduction secondary to surface IgM cross-linking on B-chronic lymphocytic leukemia cells: differential activation of the phosphatidylinositol-specific phospholipase C.
J Immunol.
1990;144:2351-2362[Abstract].
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Michel F, Merle-Beral H, Legac E, Michel A, Debrè P, Bismuth GJ.
Defective calcium response in B-chronic lymphocytic leukemia cells.
J Immunol.
1993;150:3624-3632[Abstract].
Response:
Response to surface IgM engagement in CLL
We appreciate the letter of Dr Zupo and colleagues who
like our group have studied the responsiveness of chronic
lymphocytic leukemia (CLL) cells to signals through the
antigen receptor.1,2 Those authors comment on several
aspects of our work including the reagents used, the possible existence
of CLL subsets in terms of responsiveness to signals through the B-cell
receptor (BCR) for antigen, and the biological significance of our
observations including apoptosis inhibition and induction of
antiapoptotic bcl-2 family members in stimulated CLL cells. Our data
are important because they provide the first demonstration of a
molecular pathway by which antigen extrinsic to malignant
lymphocytes can promote tumor cell survival. We agree with these investigators that the nature of the
immunoglobulin (Ig) reagent used to stimulate human B cells in culture can affect the outcome of survival or death. We utilized an
F(ab')2 preparation of polyclonal goat antibody to human
IgM heavy chains (American Qualex, San Clemente, CA) that was selected
to avoid any potential nonspecific effects of Fc-receptor engagement in human B cells. As CLL cells express Fc receptors, including the inhibitory receptor Fc RIIb (E.S., unpublished observations by RT-PCR, September 2001; Fridman et al3; and Gamberale et
al4), the discrepancy between our observations and those of
others might arise from negative signals transmitted to CLL cells upon
exposure to intact antibody preparations. Our findings, that apoptosis is inhibited upon surface IgM engagement in CLL, are entirely compatible with a tumor that expresses IgM with affinity for autologous structures. The literature supports 2 patterns of CLL responses to
stimulation through the BCR,5,6 but we did not observe 2 distinct patterns in our apoptosis studies. Rather, the differences in apoptosis inhibition among cases were a matter of degree. Although the
effects of IgM engagement on Annexin V binding were subtle in some
experiments, there was a statistically meaningful pattern of apoptosis
inhibition among all cases evaluated.2 Moreover, in later
studies in which we used an alternative method of apoptosis measurement, propidium iodide to mark the subdiploid DNA content of
cells, the differences between unstimulated and IgM-stimulated CLL
cells were greater and more evident. Other methods we used to measure
apoptosis, including evaluation of polyADP-ribose polymerase cleavage by immunoblotting and of cell morphology by fluorescence microscopy, yielded a constant picture. The clinically relevant question is not whether apoptosis inhibition occurs in CLL upon surface
IgM engagement, but what is the degree of apoptosis inhibition in cells
stimulated by real, complex antigens in vivo? The results of our in
vitro studies may underrepresent the biological effects of
antigens to which tumor cells bind in patients with disease. Finally, we stand by our findings regarding induction of
antiapoptotic genes in CLL cells stimulated by the BCR. In
particular, increases of mcl-1 at the protein level have been clear and
reproducible in every case we have examined thus far. Consistent with
our published model for the role of CD40 ligand and antigen in CLL
pathogenesis,7 induction of bcl-2, mcl-1, and bfl-1
transcripts has been greatest when both CD40 and surface IgM are
engaged. What remains to be elucidated are the precise mechanisms by
which bcl-2- type proteins including bcl-2 and mcl-1, as well as other
inhibitors of apoptosis that are overexpressed in CLL, interact, are
metabolized, and function to prevent cell death.
Elaine Schattner and Alejandro Bernal
Correspondence: Elaine Schattner, Division of Hematology and
Medical Oncology, Weill Medical College, and Immunology Program, Weill
Graduate School of Medical Sciences, Cornell University, New York, NY
10021
References
1.
Zupo S, Massara R, Dono M, et al.
Apoptosis or plasma cell differentiation of CD38-positive B-chronic lymphocytic leukemia cells induced by cross-linking of surface IgM or IgD.
Blood.
2000;95:1199-1206.
2.
Bernal A, Pastore RP, Asgary Z, et al.
Survival of leukemic B cells promoted by engagement of the antigen receptor.
Blood.
2001;98:3050-3057.
3.
Fridman WH, Mathiot C, Montcuit J, Teillaud JL.
Fc receptors, immunoglobulin-binding factors and B chronic lymphocytic leukemia.
Nouv Rev Fr Hematol.
1988;30:311-315.
4.
Gamberale R, Geffner JR, Trevani A, et al.
Immune complexes inhibit apoptosis of chronic lymphocytic leukaemia B cells.
Br J Haematol.
1999;107:870-876.
5.
Hivroz C, Geny B, Brouet JC, Grillot-Courvalin C.
Altered signal transduction secondary to surface IgM cross-linking on B-chronic lymphocytic leukemia cells: differential activation of the phosphatidylinositol-specific phospholipase C.
J Immunol.
1990;144:2351-2358[CrossRef][Medline]
[Order article via Infotrieve].
6.
Lankester AC, van Schijndel GM, van der Schoot CE, van Oers MH, van Noesel CJ, van Lier RA.
Antigen receptor nonresponsiveness in chronic lymphocytic leukemia B cells.
Blood.
1995;86:1090-1097.
7.
Schattner EJ.
CD40 ligand in CLL pathogenesis and therapy.
Leuk Lymphoma.
2000;37:461-472[Abstract/Free Full Text]
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