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Blood, 1 April 2001, Vol. 97, No. 7, pp. 2181-2183
CORRESPONDENCE
To the editor:
Flow cytometry cannot assess surface binding of perforin to
target cells
Lehmann et al1 recently reported results
suggesting that the impaired binding of perforin (PFN) to the surface
of tumor cells is associated with resistance to cytotoxic effector cell killing. Using natural killer (NK)-sensitive (K562) and -resistant (ML-2) cell lines, they found that supernatants from freeze-thawed human CD56+ NK cells (NK lysates) did not damage ML-2 but
that K562 were permeabilized, an effect inhibited by the anti-PFN
antibody (clone  G9). Using dual fluorescence analysis, the authors
then directly demonstrated that K562 cells that were permeabilized
(propidium iodide, PI+) also stained positive for PFN. The
inference was made that most permeabilized (dead) cells had PFN on
their surface. ML-2 cells, on the other hand, showed no binding of PFN
and did not undergo lysis; therefore the authors attributed this
resistance of the ML-2 line to the inability of PFN to bind to plasma
membrane of these resistant tumor cells. In our opinion, the authors overlooked a crucial control, which
evaluates whether the PFN detected by flow cytometry (FCM) represents protein that interacted with targets after membrane damage
has occurred. We hypothesize that PFN, in amounts below the level of
detection by FCM, actually permeabilizes the target cells. But due to
its charged state,2 PFN monomers in solution could then
bind nonspecifically to sites on the membrane of necrotic cells, as
well as intracellularly. To test this hypothesis, we examined the
capacity of PFN contained in granule extracts and also isolated
PFN3 to bind to detergent permeabilized
(0.01% NP-40, 37°C, 30 minutes) Jurkat cells. The cells
were then incubated with the preparations (37°C, 60 minutes), washed
with phosphate buffered saline (PBS)-2% bovine serum albumin (BSA),
and then reacted with either FITC-anti-PFN antibody (clone
G9) or an FITC-IgG2b isotype control antibody (BD
Pharmingen, San Diego, CA). After a wash step (PBS-2% BSA),
the cells were resuspended in the same buffer containing PI (5 µg/mL)
and analyzed on a FACSCALIBUR (Becton Dickinson Immunocytometry
Systems, San Jose, CA) instrument. Figure 1 shows PI reactivity versus PFN
reactivity of nonpermeabilized and permeabilized Jurkat cells. After
treatment with PFN containing YT granule extract in a
concentration range that was previously determined to minimally
permeabilize the target (300-37.5 ng/mL), only 10% of the detergent
untreated cells were present in the double positive quadrant (Figure
1G, I, K, and M, respectively). In comparison, the NP40 permeabilized
cells exposed to PFN at similar concentrations resulted in dual
positive events ranging from 96% to 75% (Figure 1H, J, L, and N). The
percentage of double positive events was comparable for PFN
concentrations ranging from 300 ng/mL to 75 ng/mL. This absence of
concentration-dependent increase in PFN reactivity further suggests
that PFN interacted nonspecifically with the detergent permeabilized
target cells. Finally, under a fluorescent microscope, target cells
possess an intracellular rather than a cell surface staining pattern
(data not shown).
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| Figure 1.
Permeabilized cells nonspecifically bind PFN.
PI reactivity (y-axis) versus PFN reactivity (x-axis) of
nonpermeabilized (left column of panels) and NP-40 permeabilized
(right) Jurkat cells. PFN untreated cells that were exposed to isotype
control mAb or anti-PFN mAb are shown respectively as panels A-B and
C-D. Similarly manipulated subsets were treated with PFN containing YT
granule extract for 1 hour (37°C) and stained with either control
antibody (E-F) or anti-PFN antibody (G-N). The concentrations of PFN
were 300 ng/mL (E-H), 150 ng/mL (I-J), 75 ng/mL (K-L), and 37.5 ng/mL
(M-N). These concentrations were estimated based on densitometric
calculations where granules from 106 YT cells yield
approximately 3 ng of PFN. The resultant dot plots have intensity of PI
reactivity on the y-axis (FL3) and PFN reactivity on the x-axis (FL1).
Gates were established based on the antibody control groups (A-D)
allowing the quadrants to be defined as follows: upper left,
PI+ PFN ; upper right, PI+
PFN+; lower left, PIPFN; and
lower right, PI PFN+. The percentage of
events in each quadrant is provided.
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If our hypothesis is invalid, then PFN should be detectable by FCM on
nonpermeabilized cells. When isolated PFN was added to targets, only
PI+, PFN+ cells were identified (data not
shown). We then repeated the experiment without Ca++ (4mM
EDTA) to minimize membrane permeabilization. Under this condition,
approximately 10%-20% of cells were found to be PFN+ and
PI (data not shown). We were unable, therefore, to
identify cells that were PFN+ and PI unless
permeabilization was blocked. In conclusion, our observations suggest that FCM only detects PFN in
treated cells after membrane permeabilization. Therefore this technique
is not suitable to accurately detect the amount of PFN that binds and
mediates membrane damage and cannot be correlated with resistance to
PFN mediated damage. An important corollary of these studies is that
the cell membrane associated PFN described by Lehman et al actually
represents primarily intracellular PFN. Our findings do not minimize
the fundamental observation reported by Lehman et al, where the results
show that tumor cell lines display varying degrees of susceptibility to
lysis when exposed to equivalent amounts of PFN. This conclusion,
however, must be based solely on the differences in PI reactivity.
Sunil S. Metkar, M. Aguilar-Santelises, Baikun Wang, and Christopher J. Froelich
Evanston Northwestern Healthcare Research
Institute IBIS Program Northwestern
University Evanston Hospital Evanston, IL 60201 e-mail:
smetkar{at}enh.org
Acknowledgments
Supported by NIH RO-1 grant # AI 44941-01A1 to C.J.F.
References
1.
Lehmann C, Zeis M, Schmitz N, Uharek L.
Impaired binding of perforin on the surface of tumor cells is a cause of target cell resistance against cytotoxic effector cells.
Blood.
2000;96:594-600[Abstract/Free Full Text].
2.
Persechini PM, Young JD.
The primary structure of the lymphocyte pore-forming protein perforin: partial amino acid sequencing and determination of isoelectric point.
Biochem Biophys Res Commun.
1988;156:740-745[CrossRef][Medline]
[Order article via Infotrieve].
3.
Froelich CJ, Turbov J, Hanna W.
Human perforin: rapid enrichment by immobilized metal affinity chromatography (IMAC) for whole cell cytotoxicity assays.
Biochem Biophys Res Commun.
1996;229:44-49[CrossRef][Medline]
[Order article via Infotrieve].
Response:
Mechanisms of perforin resistance: the differentiation between
perforin binding and perforin-mediated lysis remains difficult
Metkar et al report that previously permeabilized Jurkat cells
show an intense staining of perforin with an FITC-labeled antibody after incubation with YT granule-extract/purified perforin compared to
nonpermeabilized Jurkat cells. They hypothesize that the reason for
their observation is an unspecific intracellular binding of perforin
(due to its cationic nature) to the cell membrane of the permeabilized
target cells. Although we are convinced that the data provided by
Metkar et al represent an interesting contribution that may help to
further explain our findings published recently,1 we are
not able to follow all of their conclusions. Several points of their
interpretation of their own and our data need to be critically discussed: First, Metkar et al conclude from the observation that Jurkat
cells permeabilized by NP-40 are strongly positive for perforin after
incubation with granule extract or purified perforin that perforin
might get into the cells, once the membrane is permeabilized, and bind
unspecifically to the inner side of the cell membrane. But to our
knowledge, it is not yet clear whether perforin pores are big enough to
allow the FITC-labeled antibody to enter the cell. In the case of the
NP-40 permeabilized cells, it is obvious that the antibody might get
into the cell since the detergent will strongly disintegrate the cell
membrane (which is often used in experiments to permeabilize cells for
an intracellular antibody staining). But this has not been shown for
perforin pores: In our opinion it would be necessary to prove that an
FITC-labeled antibody is able to get into a cell by a perforin pore
(eg, by permeabilizing cells with perforin and then adding an antibody specific for intracellular proteins). Only then would it be justified to talk about "intracellular perforin" detected by the perforin antibody (as Metkar et al claim is the case in our experiments), although there is still the possibility that the antibody binds mainly
to surface bound perforin. Second, we are concerned about the fact that the experiments
Metkar et al performed were with Ca-free buffer. They justify the use
of Ca-free media by the need to minimize membrane permeabilization. But
using a buffer without Ca must result in a strongly impaired perforin
binding, as indicated by several groups that have uniformly reported
that Ca is mandatory for perforin binding (and of course subsquent
lysis); see, for example, Uellner et al.2 In our opinion,
it is not possible to obtain conclusive data on the mechanisms of
perforin-binding and perforin-mediated lysis in the absence of Ca. The
observation of Metkar et al that they were unable to find
PFN+PI cells unless permeabilization is
blocked corresponds with our hypothesis: only cells that bind perforin
and are thus lysed can become PI+. These experiments
demonstrate that it is very difficult (if not impossible) to
differentiate between perforin binding and perforin-mediated lysis, and
it is at least questionable whether these 2 events can be investigated
separately at all. Despite our concerns regarding the interpretation of
their results, we agree with Metkar et al that the proposed mechanism
could indeed be responsible for the perforin-positive staining of
PI+ target cells and should be further investigated. In our study we used the working hypothesis that
perforin-resistant tumor cells bind less perforin on their
surface than perforin-sensitive tumor cells and found evidence that
this might indeed be the case. Although Metkar et al have presented
interesting results that could lead to another explanation for the
observations we made, we think that definitive conclusions are not
possible at the present time. As we have pointed out, further
experiments are clearly necessary to finally elucidate the precise
molecular mechanisms responsible for the heretofore undetected phenomen
of tumor-cell resistance against perforin-mediated lysis. It is
important to note that the description of this phenomenon, which was
the central issue of our work, was confirmed by Metkar et al and is not
affected by the present discussion.
Christof Lehmann and Lutz Uharek
Department of Internal Medicine Christian Albrecht
University Kiel, Germany
References
1.
Lehmann C, Zeis M, Schmitz N, Uharek L.
Impaired binding of perforin on the surface of tumor cells is a cause of target cell resistance against cytotoxic effector cells.
Blood.
2000;96:594-600.
2.
Uellner R, Zvelebil MJ, Hopkins J, et al.
Perforin is activated by a proteolytic cleavage during biosynthesis which reveals a phospholipid-binding C2 domain.
EMBO J.
1997;16:7287-7296[CrossRef][Medline]
[Order article via Infotrieve].
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