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Departments of
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Pathology and
Medicine (Division of Infectious Diseases), Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Bcl-2 family comprises 
20 proteins
(bcl2s)3 that either
promote or block apoptosis (32). However, there is little information
about the expression of these proteins in inflammatory myeloid
leukocytes. Neutrophils and macrophages express Bax, a death-promoting
bcl2 (33, 34). Bax can be antagonized by protective bcl2s; however, no
protective bcl2s have previously been identified in neutrophils. In
macrophages, protective bcl2s have generally not been detected in
inflammatory or resident cells in situ. However,
Bcl-xL, a protective bcl2, can be induced in
cultured macrophages (35), and there is one report of Bcl-2 expression
in macrophages in endometriosis (36).
We have previously described a protective murine bcl2, A1, that is rapidly induced in macrophages by either LPS or GM-CSF. In addition, A1 is expressed in promyeloid cells that have been induced to differentiate into neutrophils by G-CSF (37, 38). A1 can interact with Bax (39, 40), indicating a potential mechanism for protection. We investigated A1 expression in a model of an acute inflammatory response to an intracellular protozoan pathogen, T. gondii. The results indicate that A1 is highly expressed in infiltrating macrophages and also in both normal circulating and inflammatory neutrophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice, 6–8 wk old, were obtained from The Jackson Laboratory (Bar Harbor, ME). In some experiments, female outbred CD1 mice 5–7 wk old from Charles River (Wilmington, MA) were used. For some experiments, BALB/c mice were maintained under specific pathogen-free conditions in the barrier facility maintained by the Institute for Animal Studies at Albert Einstein College of Medicine. The RH strain (Sabin) of T. gondii was maintained on human fibroblasts as described previously (41). Supernatants containing 107 tachyzoites/ml were diluted in calcium- and magnesium-free PBS (PBS-CMF). A volume of 0.4 ml containing 1 x 103 to 1 x 104 tachyzoites was injected i.p. Vehicle controls used tissue culture medium diluted in PBS-CMF. For initiation of thioglycolate peritonitis, BALB/c mice were injected i.p. with 1.0 ml of sterile 3% thioglycolate broth (Difco, Detroit, MI) aged several months.
Harvest and extraction of cells
For peritoneal lavage, mice were sacrificed by cervical dislocation, and the peritoneal cavity was washed with 4 ml of ice-cold PBS-CMF containing 0.2% BSA (low endotoxin grade; Sigma, St. Louis, MO) (PBS-A). Cellularity and extracellular tachyzoites were assessed with a hemacytometer. Blood was collected by cardiac puncture following CO2 anesthesia. Blood from either individual mice or pools of seven mice was collected onto an equal volume of ice-cold isotonic saline containing 10 U/ml heparin and 10 mM EDTA. All further steps were at 0–4°C except as indicated. Blood was underlain with Ficoll/sodium diatrizoate (density, 1.090) and centrifuged at 600 x g for 13 min, and total leukocytes were recovered from the interface. The neutrophil count in this leukocyte fraction was similar to reported values (data not shown). The interface was diluted with 5–10 vol PBS-A, centrifuged at 150 x g for 10 min, and resuspended in PBS-A. For some experiments, peritoneal cells were washed with PBS-A, brought to room temperature, and separated by centrifugation at room temperature over Histopaque, 1.077 g/ml (Sigma), as described for blood. Samples of blood leukocytes and peritoneal cells were cytocentrifuged (700 rpm, 5 min). Slides were either air dried, fixed in methanol, and stained with Diff-Quik (Dade International, Miami, FL) for differential counting or fixed in cold paraformaldehyde (4% in PBS-CMF) for 20 min, washed several times with PBS-CMF, and stored in PBS-CMF at 5°C for up to 10 days before immunostaining. Intracellular parasitization was assessed in Diff-Quik-stained preparations by examination of 500 macrophages under oil immersion. For extraction of total cellular RNA, cell suspensions were centrifuged (150 x g, 10 min), resuspended in residual volume, lysed with Trizol (Boehringer Mannheim, Indianapolis, IN), and extracted according to the manufacturer’s protocol. For extraction of total cellular protein, cell suspensions were additionally washed in PBS to remove BSA, extracted with a modification of Laemmli gel sample buffer (38), boiled for 5 min, and stored at -20°C.
Northern blot analysis
Total cellular RNA (2–4 µg) was precipitated with sodium
acetate and ethanol, separated on 1.2% agarose-formaldehyde gels, and
transferred to nylon membranes (either Hybond-N (Amersham, Arlington
Heights, IL) or Biodyne A (Life Technologies, Gaithersburg, MD)) as
previously described (42). Prehybridization, hybridization (in 50%
formamide at 42°C), and washing of filters were as described (42).
The A1 probe was an EcoRI fragment from the pBlueA1 cDNA
plasmid previously described (38). The probes for murine Bcl-2 and Bax
were kind gifts of Dr. J. C. Reed (The Burnham Institute, La
Jolla, CA). The probe for GM-CSF has been previously described (43).
RNA loading was normalized by hybridization with the murine homologue
of a 28S rRNA-derived oligonucleotide (44), used at 5-fold molar
excess. Molar excess of the 28S probe was verified by examining serial
dilutions of sample RNA (data not shown). The Bcl-2 band migrated more
slowly than 28S rRNA, corresponding to full-length Bcl-2
message
(45). Signals were quantitated by analysis with a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
Preparation of an anti-peptide antiserum
A peptide corresponding to residues 1–16 of the murine A1 sequence, with an additional C-terminal cysteine, was synthesized by the Laboratory of Macromolecular Analysis at the Albert Einstein College of Medicine. The peptide was conjugated to maleimide-activated keyhole limpet hemocyanin (Pierce, Rockford, IL) according to the manufacturer’s protocol. A rabbit antiserum against this conjugate was prepared by Anaspec (San Jose, CA). Rabbits were injected with conjugate (>50 µg peptide) + CFA and boosted at 3, 6, and 10 wk with conjugate + IFA. Bleeds were screened initially by ELISA against a peptide-BSA conjugate and then by Western blot analysis of A1-transfected COS cells. For affinity purification, the serum was diluted in Tris-buffered saline (pH 7.4) and applied to a peptide-agarose column prepared by the covalent linkage of residues 1–16 using the Sulfolink method (Pierce) according to the manufacturer’s protocol. The column was washed with Tris-buffered saline and sequentially eluted first with 3 M potassium thiocyanate and then with 0.1 M glycine (pH 2.5). Eluates were neutralized with 0.1 vol 1 M Tris-HCl (pH 7.5) and dialyzed against PBS-CMF, and their specificity was verified by analysis of Western blots containing either mock or A1-transfected COS cells (R. D. Somogyi A. Orlofsky, L. M. Weiss, and M. B. Prystowsky, manuscript in preparation). The glycine eluate (350 µg/ml) was of higher titer and was chosen for experimental use. The affinity-purified Ab is referred to as 855AP.
Western blot analysis
Protein content of lysates was assessed by the Bradford assay. Samples were separated on 15% SDS-PAGE gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA) by wet electrophoretic transfer (Bio-Rad, Richmond, CA) in 25 mM Tris, 190 mM glycine for 40 min at 100 V. The membranes were blocked with 5% dry milk + 0.1% Tween-20 in PBS (block solution) and probed with the 855AP Ab (1:100) in block solution for 1.5 h. The blot was washed 9 times in PBS-CMF + 0.5% Tween-20, incubated in horseradish peroxidase-conjugated donkey anti-rabbit antiserum (Amersham) (1:10,00 in block solution), and washed as before. Detection was by enhanced chemiluminescence (Amersham) following the supplier’s recommended protocol. Quantitation was by densitometry (Molecular Dynamics). The m.w. standards used here are prestained and therefore only approximate. By the use of unstained standards and mass spectrometry analysis of purified A1, we have determined that the m.w. of A1 agrees with the predicted value of 20 kDa (R. D. Somogyi et al., manuscript in preparation).
Immunocytochemistry
Fixed cytospin preparations were permeabilized in 0.2% Triton X-100 in PBS-CMF for 25 min. Slides were rinsed in PBS-CMF, and endogenous peroxidase was blocked by incubation in 1 mM sodium azide, 10 mM glucose, and 1 U/ml glucose oxidase (Sigma) in PBS-CMF for 1 h at 37°C (46). The slides were rinsed with PBS-CMF and blocked for 30 min in PBS-CMF containing 0.05% Triton X-100, 2% BSA, and 1% goat serum. The slides were then probed with the 855AP Ab (1:100 in the block buffer) for 2 h. The cells were washed four times with PBS-CMF + 0.05% Tween-20, and the signal was detected by the Ultrasensitive ABC method (Pierce) according to the supplier’s protocol using diaminobenzidine. For dual detection of apoptosis and A1, fixed cytospin preparations were initially probed for nicked DNA by TUNEL using a kit from Boehringer Mannheim according to the manufacturer’s protocol. The slides were then washed with PBS-CMF containing 0.05% Triton X-100 and blocked and probed with 855AP Ab, as above. The slides were washed with PBS-CMF + 0.05% Triton X-100, the blocking step was repeated, and Texas Red-X goat anti-rabbit IgG (H+L) (Molecular Probes, Eugene, OR) was applied at 10 µg/ml for 2 h. Slides were mounted using the ProLong antifade kit (Molecular Probes) and examined by epifluorescence using I3, N2.1, or G/R filters (Leica, Deerfield, IL). Images were scanned into Adobe Photoshop 5.0.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intraperitoneal infection with tachyzoites of the virulent RH
strain of T. gondii led to a vigorous inflammatory response
characterized by an abrupt increase in both granulocyte and mononuclear
cell numbers in the peritoneal cavity at 3–4 days postinfection
(p.i.). The granulocytes were predominantly neutrophils, with a
variable number of eosinophils (<20% total granulocytes (data not
shown)). The mononuclear cells were primarily macrophages, with the
proportion of lymphocytes varying between 10 and 30% (data not shown).
The leukocyte influx was accompanied by a steady increase in both
extracellular and intracellular parasites (Fig. 1
, C and D).
Infection resulted in 100% mortality at 1 wk p.i. (data not
shown).
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, a dramatic increase in A1 expression
occurred at about day 3 p.i. and was maintained for the duration
of the experiment. In comparison, the level of Bcl-2 mRNA was
unchanged. In three similar time course experiments, the maximal
increase in A1 expression ranged from 5- to 12-fold. Similar kinetics
were obtained with either outbred CD1 or BALB/c mice (data not shown).
The kinetics of A1 expression were dependent on the dose of the
organism and generally correlated with inflammation as assessed by
peritoneal cellularity (Fig. 3, but see
Discussion).
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), although the increase (2.3-fold) was
less than that seen with T. gondii infection (7.1-fold in
this experiment). Very similar expression was observed at day 3 and day
6 of thioglycolate stimulation (data not shown). Vehicle-injected
animals showed no increase in A1.
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To examine A1 protein expression in individual cells, an
affinity-purified Ab (855AP) was prepared against a peptide
corresponding to the A1 N terminus. The ability of this Ab to recognize
A1 specifically was verified by Western blot analysis (Fig. 5). A species comigrating with A1
overexpressed in COS cells was detected in both the resting and the
inflamed peritoneal cavity (Fig. 5A). The fold increase in
signal intensity in the inflamed exudate was 3.7 ± 1.0 (SE)
(n = 3). These signals were ablated by competition with
the antigenic peptide (Fig. 5B).
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. Normal resident peritoneal macrophages
were uniformly weakly positive, showing a cytoplasmic stain that had
both a diffuse and a punctate character, with some concentration of the
punctate stain in the perinuclear region (Fig. 6, A and
C). In contrast, inflammatory macrophages from infected mice
were highly variable with regard to intensity of staining, and
approximately half of the macrophages showed an intense focal
accumulation of stain in a paranuclear location (Fig. 6D).
There was no evident correlation between A1 immunostaining and the
parasitized state of the cell, as the infected and uninfected
macrophage populations each included strongly and weakly immunoreactive
cells (Fig. 6F, arrowheads) in similar proportions (data not
shown).
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E). It is difficult to
distinguish neutrophils and eosinophils with certainty among the
immunoreactive granulocytes. However, A1-negative granulocytes were
uniformly eosinophils by eosin staining (data not shown; see Fig. 7). Therefore, it can be concluded that
inflammatory neutrophils are uniformly positive, whereas eosinophils
are largely unstained.
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Next, the reactivity of the 855AP Ab with normal peripheral blood
leukocytes was assessed (Fig. 7). As with the inflammatory infiltrate,
granulocytes were mostly positive, and negative granulocytes were in
all cases eosinophils. The number of negative eosinophils was similar
to the total number of these cells determined by differential counts
(data not shown). Therefore, A1 expression appears to be a constitutive
property of neutrophils but not of eosinophils. Blood monocytes showed
only a weak stain similar to that observed in resident peritoneal
macrophages, whereas lymphocytes were either negative or occasionally
weakly positive (Fig. 7B, arrowheads). Similar results were
obtained with mice housed under specific pathogen-free conditions (data
not shown), indicating that constitutive A1 expression does not reflect
an inflammatory process.
The elevation of A1 mRNA in inflamed peritoneum represents induction in macrophages
The constitutive expression of A1 in neutrophils raises a question
regarding the earlier observation of increased A1 gene expression in
T. gondii-elicited cells: is this elevation due to
up-regulation of A1 gene expression or simply to the recruitment of
large numbers of neutrophils already expressing A1? To address this
question, we examined A1 mRNA expression in inflammatory exudates in
which the abundance of neutrophils had been either increased or
decreased by separation of the exudate on a density gradient. As shown
in Fig. 8, depletion of up to two-thirds
of the neutrophils in the exudate had little or no effect on the
relative abundance of A1 mRNA. Conversely, fractions enriched for
neutrophils showed no increase and perhaps a slight decrease in A1 mRNA
relative abundance. Finally, total peripheral blood leukocytes, despite
containing a substantial number of A1-positive neutrophils, showed only
a very low relative abundance of A1 mRNA (Fig. 8), and this level of
expression was not reduced by removal of 93% of neutrophils with a
density gradient (data not shown). These data are consistent with
previous reports of the low abundance of RNA in neutrophils (47, 48).
The results indicate that, despite intense immunostaining, neutrophils
contribute only a very minor proportion of the RNA analyzed on Northern
blots and that the observed increase in A1 mRNA primarily reflects
up-regulated expression in the macrophage lineage.
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The function of A1 potentially involves interaction with
proapoptotic bcl2s. Yeast two-hybrid analysis has shown strong physical
interaction between A1 and Bax (39, 40), a proapoptotic bcl2 expressed
in neutrophils and macrophages (33, 34). We therefore asked whether Bax
is expressed during the inflammatory response to T. gondii.
As shown in Fig. 9, inflammatory exudates
exhibit similar up-regulation of A1 and Bax mRNAs. The kinetics of Bax
up-regulation closely resemble those of A1 (data not shown). As with
A1, Bax mRNA abundance is not reduced in neutrophil-depleted exudate,
indicating that the up-regulation is likely to occur in macrophages
(Fig. 9). In addition to the expected major 1-kb species, a minor,
lower m.w. RNA was observed. The identity of this species is
unknown.
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To assess the potential involvement of A1 in the regulation of
apoptosis, we asked whether the expression of A1 was altered in
apoptotic inflammatory cells. TUNEL-stained apoptotic macrophages and
granulocytes were detectable in T. gondii-elicited exudates,
although infrequently. Dual staining of exudates for TUNEL and A1
expression indicated that apoptotic macrophages and granulocytes
occasionally contained high levels of A1 localized to the nucleoplasm,
in contrast to nonapoptotic cells in which only cytoplasmic A1 was
observed (Fig. 10). Some artifactual
staining of apoptotic nuclei was observed with secondary Ab alone (Fig. 10E), but this was less intense than that produced by the
855AP Ab (Fig. 10B). In apoptotic macrophages with partial
TUNEL staining, A1 was observed only in the cytoplasm (data not shown).
Cytoplasmic A1 remained prominent in a portion of the apoptotic
granulocytes and macrophages, so that in some cells a whole-cell
staining pattern was observed (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While our manuscript was in preparation, two reports appeared describing the expression of A1 in neutrophils. Hatakeyama et al. (50) reported RT-PCR analysis demonstrating A1 expression in murine neutrophils. Chuang et al. (51) described the occurrence in human neutrophils of mRNA encoding A1 and a second protective bcl2, Mcl-1. Consistent with our findings, human A1 mRNA was constitutively expressed in neutrophils. In addition, neutrophil A1 mRNA was up-regulated 2–4-fold by G-CSF and by LPS. The effect of these agents on mature murine neutrophils with respect to A1 mRNA and protein levels remains to be examined.
The report of Hatakeyama et al. (50) describes the existence of four A1 genes in the mouse, three of which encode full-length A1 proteins and are coexpressed in neutrophils. A1-a is the isoform previously studied with respect to antiapoptotic function (38) and against which the 855AP Ab was raised. The three isoforms are 96–97% identical overall but diverge within the N-terminal peptide used to prepare the 855AP Ab. We are currently investigating the isoform specificity of this Ab.
The increased expression of A1 mRNA elicited by T. gondii
was generally coordinate with the abrupt influx of inflammatory cells
at days 3–4 in infected mice. Consequently, the increase in A1
expression could reflect either recruitment of A1-expressing cells or
induction of A1 expression during or subsequent to extravasation. Our
results demonstrate that both of these processes occur: infiltrating
neutrophils constitutively express A1, yet the observed increase in
peritoneal cell A1 mRNA is primarily due to up-regulation in
macrophages. This conclusion is further supported by the observation
that in individual mice, A1 up-regulation is not always precisely
coordinate with increased peritoneal cellularity. For example, of the
three mice examined at day 3 in Fig. 2, only two show elevation of A1
expression, yet all three are comparable with respect to cellularity
and frequency of neutrophils (data not shown). This suggests not only
that A1 expression is induced but that this induction is subsequent to
extravasation. The induction does not appear to be triggered by
parasitization of macrophages. Nevertheless, we have observed that
total peritoneal A1 mRNA is substantially better correlated with the
frequency of intracellular parasitization than with peritoneal
cellularity (data not shown), suggesting that the signal(s) responsible
for macrophage A1 expression is related to the pathogen-driven host
response. Preliminary results show that mRNA for GM-CSF, a cytokine
known to induce A1 in macrophages, is up-regulated during T.
gondii-elicited inflammation and this up-regulation is coordinate
with A1 expression: in the experiment shown in Fig. 2, with one
exception mice showed increased expression either of both genes or of
neither gene (data not shown). The exception was an uninfected mouse
with basal A1 and elevated GM-CSF. Therefore, inflammatory cytokines
such as GM-CSF are potential mediators of A1 up-regulation.
An interesting aspect of our results is that a portion of apoptotic
leukocytes express A1 and that in some apoptotic cells A1 displays an
unusual nucleoplasmic localization. Three possible (not mutually
exclusive) hypotheses are suggested by these findings. The first is
that upon translocation from cytoplasm to nucleus, A1 no longer
functions to counteract apoptosis. The ability of A1 to inhibit
apoptosis in neutrophils was recently demonstrated using gene targeted
mice lacking the A1-a isoform (52). Studies in other cell types have
confirmed the anti-apoptotic activity of A1-a (38, 53) and A1-b
(50). However, it is possible that the functionality of A1 is affected
by altered localization or by modifications in apoptotic cells.
Relocalization of A1 may thus represent part of a proapoptotic program.
Two other bcl2s, Bax and Bcl-XL, have been shown
to alter their subcellular localization (from cytosol to membranes)
upon induction of apoptosis (54). The functional significance of
such relocalization remains unclear. The nucleoplasmic localization of
A1 is a novel feature of this Bcl-2 family member that we have
previously observed using overexpression systems (R. D. Somogyi et
al., manuscript in preparation). A second hypothesis is that
nuclear A1 retains antiapoptotic function in inflammatory leukocytes
but that apoptosis proceeds via an A1-independent pathway in these
cells. The existence of A1-independent apoptotic pathways is supported
by a recent study demonstrating that induction of A1 in human
monocytes correlates with selective resistance to a subset of apoptotic
inducers (55). The third hypothesis is that the induction of A1 in
apoptotic leukocytes (at least in macrophages, which do not uniformly
express A1 before apoptosis) occurs after the onset of apoptosis.
Recent studies have indicated that members of the TNF family that
induce apoptosis can also up-regulate antiapoptotic activity dependent
on protein synthesis (56, 57, 58). TNF- in fact up-regulates A1 in human
endothelial cells (59). However, neutrophil sensitivity to
TNF--mediated apoptosis was not enhanced by A1-a deficiency
(52).
The occurrence of apoptotic macrophages implies the existence of inflammation-associated apoptotic inducers, because this cell type normally shows little spontaneous apoptosis. The presence of proapoptotic factors is also implied by the propensity of macrophages from T. gondii-infected mice to enter apoptosis upon culture (13). Our results indicate that Bax may serve as a mediator of the effects of such apoptotic inducers in inflammatory macrophages. Nitric oxide, a molecule that is associated with host defense to T. gondii (60, 61) and that can induce apoptosis in macrophages (62, 63), has been reported to up-regulate Bax in this cell type (33). It will be of interest to determine the ability of A1 to counter the effects of nitric oxide and other potential apoptotic inducers.
Neutrophilic inflammation is often highly transient, yet in other cases, such as the peritonitis we have examined, it is maintained for many days. More sustained neutrophilic responses might result from prolonged expression of immigration signals or alternatively from delay of apoptosis, or both. Neutrophils harvested from certain inflammatory sites have been shown to have enhanced longevity in culture relative to normal peripheral blood neutrophils (10, 11, 12) (52), whereas the reverse has been reported for neutrophils elicited by thioglycolate broth (2). It is possible that these various inflammatory environments differ in their ability to maintain or enhance neutrophil A1 expression. Preliminary results indicate that A1-negative peritoneal neutrophils occur with high frequency within 8 h after thioglycolate injection (data not shown). Conversely, the enhanced longevity of proteose peptone-elicited neutrophils has been shown to be an A1-dependent phenomenon (52). The ability to examine the expression of the A1 protein in individual cells will facilitate investigation of the role of this molecule in the modulation of inflammatory reactions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Amos Orlofsky, Department of Pathology, Albert Einstein College of Medicine, F717N, 1300 Morris Park Avenue, Bronx, NY 10461.E-mail address:
3 Abbreviations used in this paper: bcl2, Bcl-2 family member; PBS-CMF, calcium- and magnesium-free PBS; PBS-A, PBS-CMF containing 0.2% BSA; p.i., postinfection.
Received for publication November 13, 1998. Accepted for publication April 8, 1999.
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References |
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or cycloheximide. J. Immunol. 154:2403.[Abstract]
T cells prevents apoptosis of macrophages and contributes to host defense in mice infected with Toxoplasma gondii. J. Immunol. 159:2375.. J. Exp. Med. 181:127.-helices of murine granulocyte-macrophage colony-stimulating factor result in a loss in bioactivity and altered glycosylation. J. Biol. Chem. 266:5333.
B activation attenuates apoptosis resistance in lymphoid cells. Blood 91:4624.B. Trends Cell Biol. 8:107.[Medline]
B activation is a critical regulator of human granulocyte apoptosis in vitro. J. Biol. Chem. 274:4309. is inhibited by the Bcl-2 family member, A1. J. Biol. Chem. 271:27201.This article has been cited by other articles:
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S. Kausalya, R. Somogyi, A. Orlofsky, and M. B. Prystowsky Requirement of A1-a for Bacillus Calmette-Guerin-Mediated Protection of Macrophages Against Nitric Oxide-Induced Apoptosis J. Immunol., April 1, 2001; 166(7): 4721 - 4727. [Abstract] [Full Text] [PDF]
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 S. Goebel, U. Gross, and C. G. K. Luder Inhibition of host cell apoptosis by Toxoplasma gondii is accompanied by reduced activation of the caspase cascade and alterations of poly(ADP-ribose) polymerase expression J. Cell Sci., January 10, 2001; 114(19): 3495 - 3505. [Abstract] [Full Text] [PDF]
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A. Villunger, L. A. O'Reilly, N. Holler, J. Adams, and A. Strasser Fas Ligand, Bcl-2, Granulocyte Colony-Stimulating Factor, and p38 Mitogen-activated Protein Kinase: Regulators of Distinct Cell Death and Survival Pathways in Granulocytes J. Exp. Med., August 28, 2000; 192(5): 647 - 658. [Abstract] [Full Text] [PDF]
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) and total extracellular
tachyzoites (C and D, filled bars).
Cytospins were prepared and counted to determine total granulocytes
(A and B, dashed line, •) and
percentage of macrophages containing parasites (C and
D, open bars; days 1–4 only). Each point represents a
minimum of three animals ± SE.
