| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
The Arthritis Center, Department of Medicine, and
Department of Microbiology, Boston University School of Medicine, Boston, MA 02118; and
Department of Environmental Health, School of Public Health, Boston University School of Medicine, Boston, MA 02118
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
FasL-expressing cells also produce microvesicles (FasL vesicle preparation (VP)) that bear FasL and display FasL function (7, 8, 9, 10). We have recently shown that the FasL VP prepared from hFasL-PA317 cells (a retroviral packaging cell line for the human FasL gene) are highly cytotoxic and capable of killing sensitive targets within a 5-h time period (9, 10). In contrast to cell-associated FasL and sFasL, the FasL VP-mediated killing of the B lymphoma target LB27.4 can be enhanced by low doses of anti-FasL mAb (NOK-1, mouse IgG1), whereas high doses of NOK-1 inhibit the activity of all three forms of FasL (10). Cross-linking of FasL VP by a low dose of NOK-1 could work by generating complexes that consist of multiple bioactive vesicles. Such complexes could interact more effectively with the target cells. Alternatively, FcR expressed by the LB27.4 cells could bind the NOK-1/FasL VP complexes and increase the extent of interaction between FasL VP and target cells. The latter explanation implies that the enhancement of FasL VP-mediated cytotoxicity depends on the subclass of anti-FasL mAb and the FcR expression level of the targets. Here, we examined six anti-FasL mAb, seven target cell lines, and FasL VP prepared from cell lines expressing either human (h) FasL or murine (m) FasL. The data presented herein establish that an FcR-dependent mechanism is responsible for the anti-FasL-mediated enhancement of FasL VP cytotoxicity. The anti-FasL mAb-mediated, FcR-dependent enhancement of FasL VP cytotoxicity represents a novel form of cytotoxicity previously unknown in the immune system. The significance, implications, and use of the novel cytotoxic mechanism are discussed.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A retroviral packaging cell line carrying the hFasL gene (hFasL-PA317) was prepared according to the method described by A. D. Miller (11). The hFasL cDNA (provided by Dr. S. Nagata, Osaka University Medical School, Japan) was cloned into pLXSN (A. D. Miller, Fred Hutchinson Cancer Research Center, Seattle, WA; accession no. M28248), and the construct was transfected into PE501 cells (obtained from A. D. Miller) with lipofectamine. The virus-laden supernatant was used to infect PA317 cells (American Type Culture Collection (ATCC), Manassas, VA) for 24 h followed by selection with G418 (0.4 mg/ml)-containing culture medium. Six cloned G418-resistant cell lines were derived. One clone was expanded and used in this study. A similarly prepared packaging cell line carrying the human cKrox gene (Krox-PA317) was used as control throughout the study (12).
The N2-mFasL cell line was prepared by electroporating the Neuro-2a tumor cell line (ATCC) with a mouse FasL gene whose metalloproteinase- sensitive site had been deleted (13). We tested 20 G418 (0.8 mg/ml)-resistant clones and found 19 of them to express strong cell-mediated cytotoxicity. A clone was expanded for use in this study. The L5-mFasL cell line, derived by transfecting L5178Y (ATCC) cells with the same mouse FasL gene, has also been described (13).
FasL VP
Cloned hFasL-PA317 and N2-mFasL cells were maintained in a 150
mm x 25 mm culture dish in 30 ml of G418-containing culture
medium. When the cells reached 
70% confluence, fresh culture medium
without G418 was used to replace the G418-containing medium.
Supernatants were collected 48 h later and centrifuged at 13,000
rpm in a Sorvall Superspeed centrifuge (Newton, CT) at 5°C for 30 min
to remove cell debris. To generate human FasL VP, the cell-free
supernatants were centrifuged for 3 h at 5°C at 25,000 rpm in a
Beckman ultracentrifuge (Beckman Coulter, Fullerton, CA) using an SW25
rotor. The pellet was suspended with culture medium to 7% of the
original volume and passed through a 0.45-µm sterile filter. Less
than 10% of cytotoxic activity was lost after the filtration, which
also defines the upper size limit of the bioactive vesicles. To
generate human sFasL, the cell-free supernatants were centrifuged for
18 h at 5°C at 25,000 rpm in a Beckman ultracentrifuge using an
SW25 rotor, and the top 80% of the supernatant was collected. The
biologic activities and the physiochemical properties of FasL VP and
sFasL have been characterized in previous studies (9, 10).
Both human FasL VP and human sFasL express cytotoxicity against LB27.4
target cells. The FasL VP have a density of 1.14–1.16 g/ml, contain
full-length FasL, and are retained by a filter (Centriprep 500;
Millipore, Burlington, MA) that excludes particles with a
molecular mass smaller than 500 kDa. In contrast, sFasL readily passes
through Centriprep 500, but is retained by a filter that excludes
particles with a molecular mass less than 50 kDa (Centricon-50;
Millipore). A previous study has shown that some sFasL exist as
aggregates of 300 kDa (14).
The same procedures were used to prepare FasL VP from N2-mFasL cells that overexpress mouse FasL lacking the metalloproteinase-sensitive site. Following ultracentrifugation, the murine FasL VP displayed strong FasL-specific cytotoxicity, and full-length FasL could be detected by Western blot using polyclonal rabbit anti-mouse FasL Abs. Cytotoxic activity was not detected in the supernatant obtained following ultracentrifugation. In addition, sFasL was not detected in the supernatant by Western blot analysis. As described in a previous study (13), Western blot analysis detected sFasL in supernatants that were prepared from a cell line overexpressing the wild-type FasL (data not shown).
Cytotoxicity assays
Target cells, LB27.4 (B lymphoma hybridoma; ATCC), LF+ (T lymphoma; Ref. 15), K31H28 T cell hybridoma (K3; Ref. 16), M59 (macrophage hybridoma; Ref. 17), FcR- A20, FcR+ A20 (kindly provided by Dr. C. Janeway, Yale University, New Haven CT; Ref. 18), or Jurkat (T lymphoma, obtained form Dr. R. Tepper, Massachussetts General Hospital, Boston, MA) were labeled with Na251CrO4 as previously described (19). Various amounts of the FasL VP were cultured with 2 x 104 target cells, in a total of 0.2 ml in each well of a 96-well plate. At 5 or 16 h after culture, supernatants were removed and counted with a gamma-scintillation counter (LKB, Turku, Finland). Use of the 5-h and 16-h cytotoxicity assays was based on the sensitivity of the various target populations under the specific assay conditions. Target cells cultured in the absence of FasL VP were used as background release. Target cells lysed with 0.5% Nonidet P-40 were used as total release, which represents 100% cell death. Cytotoxicity is expressed as percentage specific 51Cr release according to the formula: 100 x (experimental release - background release)/(total release - background release). The effect of anti-FasL mAb was studied by adding various dilutions of the mAb at the beginning of culture. The anti-human FasL mAb tested were NOK-1 (mouse IgG1; PharMingen, San Diego, CA), NOK-2 (mouse IgG2a; PharMingen), NOK-3 (mouse IgM; provided by K. Okumura) (5), G247.4 anti-FasL mAb (mouse IgG1; PharMingen), and Alf-1.2 (mouse IgG; provided by D. Kaplan, Case Western Reserve University, Cleveland, OH) (20). The isotype of Alf-1.2 has not been established. The anti-mouse FasL mAb Kay-10 was also used in these studies (mouse IgG2b; PharMingen) (21). Normal mouse isotypes were used as controls. The preparation of Fas-IgG1, a mouse Fas and human IgG1 fusion protein, has been described (16). To determine the role of mouse FcR, 2.4G2 anti-FcR mAb (rat IgG2b; PharMingen) (22) or normal rat IgG2b (PharMingen) control was added together with the anti-FasL mAb at the beginning of culture. None of these Abs display detectable cytotoxicity against various targets used in this study. All experiments were conducted in duplicate and conducted two times or more.
FcR expression
Cell surface FcR expression on target cells was determined by flow cytometry using biotin-conjugated 2.4G2 mAb. Cell-bound 2.4G2 mAb was measured with PE-streptavidin. Biotin-conjugated rat IgG2b was used as a specificity control.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We determined the ability of NOK-1 anti-human FasL mAb to
modulate the cytotoxicity mediated by cell-associated human FasL, FasL
VP, and sFasL prepared from the hFasL-PA317 cell line. Similar
preparations from the control Krox-PA317 cell line were used to
establish the specificity of these reagents (9, 10). Five
targets (LB27.4, LF+, M59, Jurkat, and K3)
frequently used in our laboratory were compared. We first tested the
effect of NOK-1 on FasL VP cytotoxicity (Fig. 1
). For each target we used a
concentration of FasL VP that induced between 40 and 60% specific
lysis of that target for the analysis. In the presence of high
concentrations of NOK-1 (1–10 µg/ml), target death induced by FasL
VP was strongly inhibited. As the NOK-1 concentration decreased, a
dose-dependent reduction of inhibition was observed. Interestingly, as
the concentration decreased further, a significant enhancement of
killing was observed with LB27.4, LF+, and M59,
but not with Jurkat and K3 target cells. The latter observation
indicates that cross-linking of FasL VP by NOK-1 is not sufficient to
enhance cytotoxicity. The "bell-shaped" enhancement
response was observed in a dose range between 1 and 100
ng/ml. Under identical conditions, no effect on cytotoxicity was
observed with isotype control Abs (not shown).
|
). The cytotoxicity of cell-associated
FasL was also determined. Both cell lines expressed strong
cell-mediated cytotoxicity (Fig. 2a). As predicted,
cytotoxic activity was not detected in the supernatant (Fig. 2b). Interestingly, FasL VP of N2-mFasL expressed strong
cytotoxicity whereas little cytotoxicity was detected in the FasL VP
prepared from L5-mFasL (Fig. 2c), suggesting variability in
the production of bioactive vesicles among different cell lines (S.
Jodo et al., unpublished observation). Therefore, we tested Kay-10 mAb,
which is specific for the mouse FasL (21), for the ability
to modulate the cytotoxic activity of FasL VP prepared from N2-mFasL
cells (Fig. 2d). We observed a similar pattern of
enhancement and inhibition with LB27.4 target cells. Moreover, the dose
ranges that respectively enhanced and inhibited the cytotoxicity were
similar to that described in Fig. 1
. Furthermore, inhibition but not
enhancement was observed when cytotoxicity was assessed with Jurkat
target cells. Taken together, these studies consistently demonstrate
enhancement of FasL VP-mediated cytotoxicity by a variety of
FasL-specific Abs with several cellular targets and several FasL VP
preparations in multiple experiments.
|
In an early study, we examined the ability of NOK-1 to modulate
the cytotoxicity of cell-associated FasL and sFasL against LB27.4
target cells (10). We found that 1–100 ng/ml NOK-1 weakly
enhanced the cytotoxicity of hFasL-PA317 cells. A similar result was
obtained with N2-mFasL (data not shown). Because both cell lines
efficiently produce FasL-expressing bioactive vesicles, the weak
enhancement may not be the property of cell-associated FasL. We have
examined a panel of FasL-expressing cell lines and found two (hFasL-3T3
and L5-mFasL) that did not produce a significant level of FasL VP
during the 5-h cytotoxicity assay. Therefore, the ability of NOK-1 and
Kay-10 to modulate the cytotoxic activity of hFasL-3T3 and L5-mFasL was
determined (Fig. 3). The results were
similar in both cases. Inhibition of cytotoxicity was observed with mAb
concentration between 1 and 10 µg/ml. In contrast to the enhancement
observed with FasL VP, the anti-FasL mAb, in a range between 1 and
100 ng/ml, did not enhance the cell-mediated cytotoxicity against
LB27.4 target cells. In addition, as previously described, inhibition
(at the high dose range) but no enhancement (at the low dose range) of
human sFasL cytotoxicity was observed with NOK-1 mAb (Fig. 3). These
results indicate that only FasL VP have the capacity to work in concert
with anti-FasL mAb and result in enhancement of cytotoxicity.
|
Because previous studies have shown that Jurkat cells do not
express the FcR (23), we hypothesized that the enhancement
of FasL VP cytotoxicity by anti-FasL mAb was mediated by Fc/FcR
interactions that focus bioactive vesicles on the target cells. Whether
the enhancement of cytotoxicity correlated with target FcR expression
was determined by fluorescent staining using the FITC-conjugated 2.4G2
anti-FcR mAb. The results showed a specific and strong staining of
LB27.4, LF+, and M59 cells (target populations
that exhibit "enhanceable cytotoxicity"). In contrast, staining of
K3 cells, the "nonenhanceable target population," was extremely
weak (Fig. 4a). To further
demonstrate that the enhancement of FasL VP cytotoxicity depends on FcR
expression, we compared the effect of NOK-1 mAb on the cytotoxicity of
FasL VP against FcR+ A20 and
FcR- A20 target populations. As shown in Fig. 4b, FasL VP killed both targets, and the sensitivity of the
two targets was comparable. In the presence of 10 ng/ml NOK-1 mAb,
killing of FcR+ A20 was significantly enhanced.
The enhancement was observed over a wide range of the FasL VP
concentrations examined. In contrast, killing of the
FcR- A20 target by FasL VP was inhibited (Fig. 4b). We also determined the effect of various doses of NOK-1
mAb on FasL VP cytotoxicity. Although a dose-dependent inhibition was
observed with the FcR- A20 target, the killing
of FcR+ A20 was enhanced by low doses of NOK-1
mAb and inhibited by high doses of NOK-1 mAb (Fig. 4c).
|
To directly establish the role of target FcR in the enhancement of
FasL VP-mediated cytotoxicity, we used the 2.4G2 anti-FcR mAb to
determine whether enhancement of cytotoxicity could be blocked (Fig. 5). As shown in Fig. 5a, 10
ng/ml of NOK-1 mAb increased the FasL VP-mediated cytotoxicity against
51Cr-labeled LB27.4 cells from 19 to 72%. The
presence of 2.4G2 mAb inhibited this enhancement of cytotoxicity in a
dose-dependent manner. The enhancement was completely blocked by 100
ng/ml of 2.4G2 mAb, while a significant blocking was still observed
with 2.4G2 mAb at a concentration of 10 ng/ml. No effect was observed
with a normal rat IgG2b control mAb. Next, we determined the ability of
various doses of NOK-1 to modulate the cytotoxicity of FasL VP in the
presence of excess 2.4G2 mAb (500 ng/ml). The results show that the
enhancing effect of NOK-1 mAb was completely blocked, so that the
entire peak of enhancement was eliminated. In contrast, the ability of
NOK-1 mAb to inhibit cytotoxicity was not blocked (Fig. 5b).
In addition to eliminating the enhancing effect of NOK-1 mAb, 2.4G2
appeared to facilitate the ability of NOK-1 mAb to inhibit
cytotoxicity. This is shown by the ability of NOK-1 in the low dose
range (1–100 ng/ml) to effectively inhibit cytotoxicity in the
presence of 2.4G2 mAb.
|
We tested a panel of anti-FasL mAb to determine the effect of
anti-FasL mAb subclass and specificity on FasL VP cytotoxicity
against the FcR+ LB27.4 target cells (Fig. 6). The same response pattern, i.e.,
enhancement with low concentrations and inhibition with high
concentrations, was observed with NOK-2 and Alf-1.2 mAb, as had been
seen with NOK-1 mAb (Fig. 1). An IgM anti-FasL mAb (NOK-3) did not
enhance cytotoxicity at the low dose range but did inhibit cytotoxicity
at the high dose range, consistent with the fact that the enhancement
was dependent on the Fc of the IgG and not the IgM class of the
FasL-specific mAb. Interestingly, the IgG1 mAb, G247-4, enhanced the
FasL VP-mediated cytotoxicity even at the high dose range. Under
identical conditions, G247-4 inhibited the FasL VP-mediated
cytotoxicity against Jurkat target cells (Fig. 7a). Next, we determined
whether the G247-4-mediated enhancement of FasL VP cytotoxicity against
LB27.4 target cells could be blocked by 2.4G2 mAb. The presence of
2.4G2 not only eliminated the enhancement but also allowed G247-4 to
inhibit the cytotoxicity against LB27.4 (Fig. 7b). The
conversion from enhancement to inhibition was not observed with rat
IgG2b control. The presence of 2.4G2 did not influence the ability of
G247-4 to block the killing of Jurkat cells (Fig. 7a). It
was noted that the ability of G247-4 to inhibit cytotoxicity was weaker
than the other mAb (compare with Fig. 6). G247-4 binds to a region near
the "self-association" site (14). Such binding may not
be as effective as binding to the FasL binding site for blocking
cytotoxicity. It is also possible that G247-4 has a low affinity for
FasL. The data nevertheless demonstrate that both the subclass and the
fine specificity of the anti-FasL mAb are important parameters for
modulating the cytotoxicity of FasL VP.
|
|
The Fas moiety on Fas-IgG1 fusion protein is a dimer rather than
the trimer of natural Fas. Therefore, it interacts with the FasL
binding site with a lower affinity than cell-associated Fas. Fas-IgG1
could be used to determine unambiguously whether enhancement of FasL
VP-mediated cytotoxicity could be achieved with a molecule that binds
to FcR (through IgG1 Fc) and to the FasL binding site (through the Fas
dimer). Therefore, we determined the effect of Fas-IgG1 on the
cytotoxicity mediated by FasL VP against Jurkat and LB27.4 targets. The
results shown in Fig. 8 indicate that
Fas-IgG1 is a strong inhibitor of FasL VP when cytotoxicity was
assessed on Jurkat target cells. In contrast, strong enhancement but no
inhibition of cytotoxicity was observed with LB27.4 target cells. Like
G247-4, enhancement of cytotoxicity was observed with high
concentrations of Fas-IgG1. Moreover, 2.4G2, but not a control Ab,
converted the Fas-IgG1-mediated enhancement of cytotoxicity to
inhibition of cytotoxicity. Cytotoxicity was inhibited more than 50%
with 100 ng/ml of Fas-IgG1, and complete inhibition of cytotoxicity was
obtained with 1–10 µg/ml of Fas-IgG1. Taken together, the data
demonstrate that FcR-dependent enhancement of FasL VP-mediated
cytotoxicity significantly reduces the inhibitory ability of Fas-IgG1,
even though the Fas-IgG1 interacts directly with FasL binding
sites.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The fact that enhancement of cytotoxicity was observed only with FasL VP and at a relatively low concentration of anti-FasL mAb (1–100 ng/ml) indicates that this phenomenon depends on the unique physical properties of the FasL VP and on a critical concentration of anti-FasL that facilitates association between FasL VP and the target population but does not block all FasL binding sites. The small size of FasL VP as microvesicles (<0.45 µm in diameter) in comparison to cells suggests that binding of a few anti-FasL mAb molecules would allow the focusing of FasL VP to LB27.4 through Fc/FcR binding. Increasing the concentration of the anti-FasL mAb blocks most or all FasL binding sites and results in inhibition of cytotoxicity.
There may be several possible reasons why mAb-mediated enhancement was not observed with cell-associated FasL. First, effector cells may use other interaction molecules such as integrins to facilitate their engagement with targets. Second, the interaction between vesicles and cells may rely more on Brownian movement. Brownian movement, coupled with the FcR-mediated focusing onto target cells, could provide faster and more effective Fas/FasL interaction in the latter case. Third, the amount of anti-FasL mAb needed to link an effector cell with a target may be so large that it is within the dose range that inhibits FasL cytotoxicity.
The results obtained with sFasL are consistent with the idea that
multivalency of the FasL is a critical factor. It has been suggested
that recombinant sFasL, consisting of the entire extracellular domain,
could exist in a multimeric form as 300-kDa aggregates, or about
four trimeric units of FasL (14). We have shown that
natural sFasL is heterogeneous and can be separated by filtration into
two fractions, one with molecular masses between 100 and 500 kDa and
the other between 50 and 100 kDa (10). Thus, sFasL may
have limited valency. If so, it is not surprising that binding of NOK-1
anti-FasL mAb to natural sFasL could not effectively focus a
significant level of "noninhibited" sFasL on LB27.4 to enhance
cytotoxicity. In addition to NOK-1, inhibition but not enhancement of
sFasL cytotoxicity was observed with NOK-2, NOK-3, and Alf-1.2 (data
not shown). These results suggest that FasL VP particles are more
multivalent than sFasL and display the appropriate physical form
necessary to enhance cytotoxicity against FcR+
targets.
Once bound to FcR on target cells, there are two possible mechanisms whereby FasL VP might deliver its apoptotic signal. The focused FasL VP could either cross-link Fas on the very cell to which they attached or they could cross-link Fas on a neighboring bystander. In preliminary studies, enhancement by NOK-1 mAb was observed when cytotoxicity was conducted with 51Cr-labeled FcR+ A20 in the presence of either unlabeled FcR+ A20 or unlabeled FcR- A20 cells. Enhancement was not observed when cytotoxicity was conducted with 51Cr-labeled FcR- A20 in the presence of unlabeled FcR+ A20. These observations strongly suggest that NOK-1 mAb only enhances FasL VP-mediated cytotoxicity on target cells with functional FcR and that the bound FasL VP does not enhance killing of neighboring bystanders. FcR may mediate enhancement simply by binding the Fc of NOK-1 mAb and thereby increasing the effective concentration of FasL VP at the cell surface. Alternatively, cross-linking FcR may transduce a signal that sensitizes the Fas-mediated death pathway. However, FcR mediates an inhibitory signal in B cells but an activation signal in T cells and macrophages (24), yet enhancement of cytotoxicity is observed in FcR+ target independent of this dichotomy. In addition, enhancement by NOK-1 mAb was not observed with sFasL and cell-associated FasL. Furthermore, a high dose of genestein (0.2 mM) known to inhibit protein tyrosine phosphorylation did not affect FasL VP cytotoxicity against LB27.4 target cells and had no effect on the enhancement of FasL VP cytotoxicity by NOK-1 mAb (data not shown).
Distinct protocols were used to generate the panel of anti-FasL mAb used in this study. The fine specificity of some of these mAb has been analyzed (14). NOK-1 and G247-4 are thought to recognize the FasL binding site (the C-terminal region) and a segment near the "self-association" site (amino acid residues 103 to 136, nonbinding site), respectively (14). The observation that high concentrations of G247-4 enhance the cytotoxicity of FasL VP suggests that the enhancement of FasL VP activity is dependent on the fine specificity or the affinity of the anti-FasL mAb. The latter interpretation was supported by similar results obtained with Fas-IgG1, which displays Fas as dimer and interacts with FasL binding site with a lower affinity than natural Fas. It should be noted, however, that both binding of G247-4 to the non-FasL binding site and binding of Fas-IgG1 to the FasL binding site resulted in inhibition of cytotoxicity if the FcR binding was blocked by 2.4G2 or if cytotoxicity was assayed on the FcR- Jurkat cells. These data suggest that reagents specific for either binding site or nonbinding site epitopes of FasL possess the ability to enhance and inhibit cytotoxicity of FasL VP.
Although we could not demonstrate a significant enhancing effect of
NOK-1 anti-FasL mAb on the cytotoxicity of cell-associated FasL,
several observations suggest that FcR-mediated interactions reduce the
ability of anti-FasL mAb to efficiently block cytotoxicity. First,
the amount of the NOK-1 anti-FasL mAb needed to inhibit the killing
of Jurkat cells was less than that needed to inhibit the killing of
LB27.4 target (Figs. 1 and 3). Second, the amount of NOK-1
anti-FasL mAb needed to inhibit the killing of LB27.4 was
significantly reduced in the presence of 2.4G2 (compare Fig. 1 and Fig. 5). Third, 2.4G2 mAb converted enhancement to inhibition when
cytotoxicity assays were conducted in the presence of G247-4, i.e., the
inhibition was observed in the dose range that enhanced cytotoxicity in
the absence of 2.4G2 (Fig. 7b). Finally, we observed that
the ability of NOK-1 anti-FasL mAb to inhibit the killing of LB27.4
by hFasL-3T3 was enhanced in the presence of 2.4G2 mAb (data not
shown).
The demonstration of Ab-mediated, FcR-dependent enhancement of
cytotoxicity represents a newly identified cytotoxic mechanism of the
immune system. In addition to understanding the properties of the
FasL-expressing bioactive vesicles, sFasL and cell-associated FasL, the
enhancement of FasL VP-mediated cytotoxicity offers a very sensitive
assay for detecting anti-FasL mAb. We have used this assay to test
a panel of IgG anti-FasL mAb. Three mAb were able to enhance
cytotoxicity at 0.01–1 ng/ml. There may be a physiological
significance to this observation. It has been shown that a significant
percentage (30%) of sera from patients with systemic lupus
erythematosus contains autoantibodies against FasL (25).
It has also been shown that FasL bioactive vesicles can be produced
from activated T cells (8), tumor cells,
(26), and a number of transfected cell lines
overexpressing FasL (9, 10). In view of the powerful
enhancing ability of anti-FasL mAb (below nanogram/milliliter
level), it is highly possible that anti-FasL-mediated enhancement
of cytotoxicity against FcR+ targets could take
place in patients with systemic lupus erythematosus or other autoimmune
diseases.
Finally, our demonstration that the IgG anti-FasL mAb can focus
FasL VP onto target cells through Fc/FcR interactions is reminiscent of
several earlier studies of viral replication in
FcR+ cells (27, 28, 29). Peiris et al.
reported Ab-dependent enhancement of virus replication in
FcR+ cells, and they demonstrated that the
enhancement was blocked by anti-FcR Ab (27). Homsy et
al. have shown that anti-HIV Ab enhance HIV infection in human
cells, and the enhancement of HIV infection was mediated through FcR
and not CD4 (28). It is also known that neutralized Dengue
virus displays residual infectivity mediated through high-affinity IgG
FcR (29). Our study extends these observations from viral
replication to the expression of apoptotic function of the FasL VP. In
this respect, the FcR-dependent, Ab-mediated interaction between
retroviral particles and target cells may be exploited for the
enhancement of gene transfer in FcR+ cells. Our
study also raises concerns on the potential effects of IgG/FcR
interactions in using IgG fusion proteins and mAb (e.g., anti-FasL,
anti-TNF-
, etc.) as modulating agents in general. More studies
are needed to determine the physiological significance of this
phenomenon and how to exploit the Ab-mediated, FcR-dependent enhancing
effect on bioactive vesicles and virus for practical use.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Shyr-Te Ju, The Arthritis Center, K508, Boston University School of Medicine, 71 East Concord Street, Boston, MA 02118.
3 Abbreviations used in this paper: FasL, Fas ligand; VP, vesicle preparation; sFasL, soluble FasL; h, human; m, murine.
Received for publication March 13, 2000. Accepted for publication August 14, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RIII (CD16) is mediated through the chain. J. Exp. Med. 175:1381.RI (CD64) on residual infectivity of neutralized Dengue virus. Virology 260:84.[Medline]This article has been cited by other articles:
|
|
 |
|
  X. Li, Y.-H. Liu, Y.-P. Zhang, S. Zhang, X. Pu, T. A. Gardner, M.-H. Jeng, and C. Kao Fas Ligand Delivery by a Prostate-Restricted Replicative Adenovirus Enhances Safety and Antitumor Efficacy Clin. Cancer Res., September 15, 2007; 13(18): 5463 - 5473. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Zuccato, E. J. Blott, O. Holt, S. Sigismund, M. Shaw, G. Bossi, and G. M. Griffiths Sorting of Fas ligand to secretory lysosomes is regulated by mono-ubiquitylation and phosphorylation J. Cell Sci., January 1, 2007; 120(1): 191 - 199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Gregory, S. Koh, E. Huang, R. R. Saff, A. Marshak-Rothstein, S. Mukai, and B. R. Ksander A Novel Treatment for Ocular Tumors Using Membrane FasL Vesicles to Activate Innate Immunity and Terminate Immune Privilege Invest. Ophthalmol. Vis. Sci., July 1, 2005; 46(7): 2495 - 2502. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Xiao, X. Zhang, K. K. Mann, S. Jodo, L. Li, W. N. Jarjour, A. Marshak-Rothstein, D. H. Sherr, and S.-T. Ju Changes in sensitivity of peripheral lymphocytes of autoimmune gld mice to FasL-mediated apoptosis reveal a mechanism for the preferential deletion of CD4-CD8-B220+ T cells Int. Immunol., May 1, 2004; 16(5): 759 - 766. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. M. Abrahams, S. L. Straszewski-Chavez, S. Guller, and G. Mor First trimester trophoblast cells secrete Fas ligand which induces immune cell apoptosis Mol. Hum. Reprod., January 1, 2004; 10(1): 55 - 63. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. M. Abrahams, S. L. Straszewski, M. Kamsteeg, B. Hanczaruk, P. E. Schwartz, T. J. Rutherford, and G. Mor Epithelial Ovarian Cancer Cells Secrete Functional Fas Ligand Cancer Res., September 1, 2003; 63(17): 5573 - 5581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Xu, A. J. Szalai, T. Zhou, K. R. Zinn, T. R. Chaudhuri, X. Li, W. J. Koopman, and R. P. Kimberly Fc{gamma}Rs Modulate Cytotoxicity of Anti-Fas Antibodies: Implications for Agonistic Antibody-Based Therapeutics J. Immunol., July 15, 2003; 171(2): 562 - 568. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Jodo, J. T. Kung, S. Xiao, D. V. Chan, S. Kobayashi, M. Tateno, R. Lafyatis, and S.-T. Ju Anti-CD95-induced Lethality Requires Radioresistant Fcgamma RII+ Cells. A NOVEL MECHANISM FOR FULMINANT HEPATIC FAILURE J. Biol. Chem., February 21, 2003; 278(9): 7553 - 7557. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Xiao, S. Jodo, S.-s. J. Sung, A. Marshak-Rothstein, and S.-T. Ju A Novel Signaling Mechanism for Soluble CD95 Ligand. SYNERGY WITH ANTI-CD95 MONOCLONAL ANTIBODIES FOR APOPTOSIS AND NF-kappa B NUCLEAR TRANSLOCATION J. Biol. Chem., December 20, 2002; 277(52): 50907 - 50913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Hohlbaum, M. S. Gregory, S.-T. Ju, and A. Marshak-Rothstein Fas Ligand Engagement of Resident Peritoneal Macrophages In Vivo Induces Apoptosis and the Production of Neutrophil Chemotactic Factors J. Immunol., December 1, 2001; 167(11): 6217 - 6224. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Jodo, S. Xiao, A. Hohlbaum, D. Strehlow, A. Marshak-Rothstein, and S.-T. Ju Apoptosis-inducing Membrane Vesicles. A NOVEL AGENT WITH UNIQUE PROPERTIES J. Biol. Chem., October 19, 2001; 276(43): 39938 - 39944. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
