Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1876-1877
CORRESPONDENCE
 |
Letter |
To the editor:
Correction of the PNH defect by GPI protein transfer: still
an open question
Sloand et al1 report that in human red blood cells
(RBCs) the paroxysmal nocturnal hemoglobinuria (PNH) defect can be
corrected by transfer of glycosylphosphatidylinositol (GPI)-anchored
proteins to GPI-deficient cells. It has been demonstrated before by a
number of authors that cell-to-cell transfer of GPI-anchored proteins can occur, and I have no doubt that a similar event also takes place
between erythrocytes and microvesicles enriched in GPI-anchored proteins. However, in my opinion, it can be questioned whether the
experiments performed by Sloand et al1 warrant the
conclusions that were drawn.
In Figure 1B the authors show an immunoblot using a monoclonal antibody
(mAb) against CD55. This antibody reacts with more than a dozen bands
(see lanes 1, 2), making it surprising that the authors are able to
identify CD55 among many other bands of similar intensities in lane 3 of the same figure. The antibody apparently reacts with such a large
number of proteins that the immunoblot shown is by no means a proof for
the presence of CD55 in the sample
a result that the authors rely on
in a number of subsequent experiments in the paper. Such a broad
reactivity of a monoclonal antibody would prompt me to question its
specificity or check the methodology for immunoblotting. Similarly, in
Figure 1C the authors show a blot with an mAb against CD59 that reacts with 2 bands of similar intensities. Although in this case the identification of CD59 may not give rise to much criticism, one should
be aware that the same antibody is used throughout the paper in flow
cytometry experiments, and it is not clear with what it may react in
these experiments. The situation is even more worrisome in the case of
the mAb against CD55, which is also used in their flow cytometric analyses.
Even more importantly, the authors claim that in order for a
transfer of GPI-anchored proteins to RBCs to occur, the GPI anchors must be intact. This conclusion is based solely on their observation that treatment of the material containing the GPI-anchored proteins with bacterial phosphatidylinositol-specific phospholipase C
(PI-PLC) abolished transfer. Interestingly, however, it is well
known from the literature that the GPI anchors of human RBC
proteins are substituted with a fatty acyl chain on the
inositol,2-5 which renders the structures
insensitive to cleavage by PI-PLC.2,5 A careful
study of the literature would have revealed this fact. Thus, their
observation that transfer of proteins to RBCs was decreased after
PI-PLC treatment cannot be explained by GPI-anchor hydrolysis, and I
wonder what might have caused the altered findings.
Although I think that the authors are probably right that GPI-anchored
proteins readily transfer from microvesicles and possibly other sources
to RBCs, I doubt that the publication proves the point. Clearly, more
experiments and truly specific antibodies are required to support the
authors' claims. I strongly believe that such studies should be
carried out because the conceptif successfulmay indeed have
important consequences for therapy of PNH.
I would like to thank Isabel Roditi and the editors of Blood
for their helpful comments.
Peter Bütikofer
Institute of Biochemistry & Molecular Biology, University of Bern, Bern, Switzerland
| |
References |
1.
Sloand EM, Maciejewski JP, Dunn D, et al.
Correction of the PNH defect by GPI-anchored protein transfer.
Blood.
1998;92:4439-4445[Abstract/Free Full Text].
2.
Roberts WL, Myher JJ, Kuksis A, Low MG, Rosenberry TL.
Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase.
J Biol Chem.
1988;263:18766-18775[Abstract/Free Full Text].
3.
Selvaraj P, Dustin ML, Silber R, Low MG, Springer TA.
Deficiency of lymphocyte function-associated antigen 3 in paroxysmal nocturnal hemoglobinuria.
J Exp Med.
1987;166:1011-1025[Abstract/Free Full Text].
4.
Ratnoff WD, Knez JJ, Prince GM, Medof ME.
Structural properties of the glycoplasmanylinositol anchor structure of the complement membrane attack complex inhibitor CD59 [abstract].
Blood.
1990;76:389a.
5.
Walter EI, Roberts WL, Rosenberry TL, Ratnoff WD, Medof ME.
Structural basis for variations in the sensitivity of human decay accelerating factor to phosphatidylinositol-specific phospholipase C cleavage.
J Immunol.
1990;144:1030-1036[Abstract].
Response:
Strong evidence for correction of the PNH
defect
In an article recently published in Blood1
we demonstrated that GPI-linked proteins can be passively transferred
from cell to cell and be incorporated into the membrane as exemplified
using decay accelerating factor (DAF) and membrane inhibitor
of reactive lysis (MIRL). Both of these GPI-linked
proteins are well characterized on a molecular and functional level.
Our results are in agreement with other studies in vitro and
in vivo, including one by the author of the letter,2 who
demonstrated that exogenous GPI-linked molecules can incorporate into
human erythrocyte membrane.
With regard to Dr Bütikofer's criticisms of our manuscript, we
make the following comments:
1. Different CD55 and CD59 antibodies were used for immunoblotting and
for flow cytometry. The antibody used for immunoblotting was selected
because it had been previously tested for this application by the
commercial provider. Immunoblot results and flow cytometry data are not
linked, but rather are confirmatory of each other. Immunoblots were
developed using mouse CD55 and CD59 mAb and using the APAAP method
(antimouse IgG-AP conjugate and anti-AP IgG-AP conjugate). Multiple
bands were obtained for both CD55 and CD59; all immunoblots contained
the desired band, however. Immunoblots of microvesicle preparations
obtained from patients with PNH lacked the specific bands or expressed
them very faintly when equal protein loads were applied to the gel. The
two bands obtained for CD59 were of 19 and 36 kd molecular weight. 19 kd is the molecular weight of CD59. The 36 kd band corresponds to CD59
dimer, as previously described in the literature.3,4
Indeed, dimeric CD59 is the primary form on cell membranes. Bands
obtained for CD55 were of 60 kd, 38 kd, and 55 kd molecular weight. The
60 kd band represents CD55 (DAF-A), while additional forms of CD55 have
been described at 55 kd (DAF-B) and 43 kd (precursor
form5). Immunoblotting was performed on the specimens to
document the presence of GPI-linked molecules in the eluate,
microvesicles, and high-density lipoprotein (HDL). Flow cytometry was
also used, and produced compatible results, with both microvesicle
preparations and intact negative cells where an increase in
specific fluorescence after experimental transfer was detected.
2. The flow cytometric data was very clear: multiple antibodies from
different companies (Caltag, Burlingame, CA;
Pharminogen, San Diego, CA; R&D, Minneapolis, MN) as well as different
lots from the same company produced similar results; the antibodies have been used in reported studies by others. We chose to present data
using the Caltag antibody, but all results for all antibodies were
similar. The specificity of antibodies was confirmed in different assays (eg, after phospholipase C treatment to decrease fluorescence for CD55 and CD59). In addition, cell lines known to be negative for
GPI-linked proteins (Ramos cell lines, PIG-A-negative EBV transformed
cell lines) did not react with these antibodies and cell lines known to
express GPI-linked proteins (EBV-transformed cells) were strongly
positive. CD55 and CD59 staining of blood cells has become an accepted
standard for the diagnosis of PNH and the test is now available commercially.
3. Our article described elimination of transfer after the red cell
eluate (obtained from outdated red cell components) and an HDL were
exposed to PI-PLC. The exact form in which CD55 and CD59 are present in
this preparation is unknown, as is the sensitivity to PI-PLC. However,
treatment of red cells by phospholipase C is known to release
GPI-linked molecules from red cells,6,7 although red cells
are more resistant to digestion than are lymphocytes or monocytes. In
our experience, we obtain decreases in CD55 and CD59 fluorescence when
red cells are incubated for 1 hour at 37°C at a concentration of 1 unit/mL, as described in the article.
All data presented in the article provide strong evidence for our
conclusions. We appreciate the author's concerns regarding the
necessity to validate the data but believe that they are unfounded, as
meticulous attention was given to ensuring that antibodies were
specific for CD55 and CD59, and that results using different techniques
were congruent. I am pleased that Dr. Bütikofer agrees with us
regarding the basic importance of the study concepts as well as the results.
We apologize to Dr Bütikofer for not citing his work, but
our manuscript was submitted to Blood immediately
prior to his publication.
E. Sloand
J. Maciejewski
D. Dunn
J. Moss
N. Young
National Heart, Lung, and Blood Institute, National Institutes
of Health, Bethesda, MD
| |
References |
1.
Sloand EM, Maciejewski JP, Dunn D, et al.
Correction of the PNH defect by GPI-anchored protein transfer.
Blood.
1998;92:4439-4445.
2.
Civenni G, Test ST, Brodbeck U, Bütikofer P.
In vitro incorporation of GPIanchored proteins into human erythrocytes and their fate in the membrane.
Blood.
1998;91:1784-1792[Abstract/Free Full Text].
3.
Hatanaka M, Seya T, Miyagawa S, et al.
Cellular distribution of a GPIanchored complement regulatory protein CD59: homodimerization on the surface of HeLa and CD59-transfected CHO cells.
J Biochem (Tokyo).
1998;123:579-586[Abstract/Free Full Text].
4.
Nickells MW, Alvarez JI, Lublin DM, Atkinson JP.
Characterization of DAF-2, a high molecular weight form of decay-accelerating factor (DAF; CD55), as a covalently cross-linked dimer of DAF-1.
J. Immunol.
1994;152:676-685[Abstract].
5.
Seya T, Farries T, Nickells M, Atkinson JP.
Additional forms of human decay-accelerating factor (DAF).
J. Immunol.
1987;139:1260-1267[Abstract].
6.
Davitz MA, Low MG, Nussenzweig V.
Release of decay-accelerating factor (DAF) from the cell membrane by phosphatidylinositol-specific phospholipase C (PIPLC). Selective modification of complement regulatory protein.
J. Exp. Med.
1986;163:1150-1161[Abstract/Free Full Text].
7.
Holguin MH, Wilcox LA, Bernshaw NJ, Rosse WF, Parker CJ.
Erythrocyte membrane inhibitor of reactive lysis: effects of phosphatidylinositol-specific phospholipase C on the isolated and cell-associated protein.
Blood
1990;75:284-289[Abstract/Free Full Text].
