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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1362-1372
Increased Mucosal B-Lymphocyte Apoptosis During Polymicrobial Sepsis
Is a Fas Ligand But Not an Endotoxin-Mediated Process
By
Alfred Ayala,
Ying Xin Xu,
Carol A. Ayala,
Diane E. Sonefeld,
Shannon M. Karr,
Tracy A. Evans, and
Irshad H. Chaudry
From the Center for Surgical Research and Department of Surgery,
Brown University School of Medicine and Rhode Island Hospital,
Providence, RI.
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ABSTRACT |
Sepsis is reported to induce an increase in the rate of apoptosis
(Ao), in immature lymphoid cells residing in hematopoietic tissues such as the thymus and bone marrow. Alternatively, secondary lymphoid tissue, such as the spleen exhibit little innate
(unstimulated) Ao. However, it is unknown whether or not
polymicrobial sepsis has any effects on the frequency of Ao
in mucosal lymphoid tissue and what, if any, are the functional
consequences of such a change. To assess this, Peyer's patch cells
were harvested from C3H/HeN (endotoxin-sensitive) mice killed 12 or 24 hours after the onset of polymicrobial sepsis (cecal ligation and
puncture [CLP]). The results indicate that the percentage of cells
that were Ao+ as determined by flow cytometry were
markedly increased at 24 hours, but not at 12 hours post-CLP. This
correlates well with evidence of increased DNA fragmentation as well as
histological changes observed both at a light and transmission electron
microscopic level of the Peyer's patch Ao. Phenotypically,
these changes were restricted to the B220+ (B-cell)
population that also exhibited a marked increase of Fas/Apo-1 antigen
expression. The functional consequence of this increased apoptosis
appears to be associated with the endogenous stimulation (activation)
of IgA production by mucosal B lymphocytes and increased nuclear c-Rel
expression. Furthermore, we found that Peyer's patch lymphocytes
isolated from C3H/HeJ-Faslgld
(endotoxin-tolerant/Fas ligand- [FasL] deficient) as opposed to
C3H/HeJ (endotoxin-tolerant) inbred mice did not exhibit increased Ao after CLP. These findings indicate that increased B-cell
Ao appears to be a FasL-Fas antigen-mediated process, but
is not due to endotoxin sensitivity. In conclusion, we speculate that the increased Fas-associated apoptosis detected in mucosal B cells (as
opposed to splenic or bone marrow B cells) may be due to increased luminal antigens other than endotoxin, released due to gut barrier integrity breakdown during sepsis.
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INTRODUCTION |
PROGRAMMED CELL DEATH (PCD), is a process
by which cells undergo a form of non-necrotic cellular suicide. As
opposed to necrotic cell death (typically produced by exposure to a
variety of noxious agents), PCD by definition is dependent on the de
novo synthesis of specific genes that initiate the cellular suicide
program in response to stimuli.1,2 The induction of PCD in
immune cells is typically evidenced by a pathological process referred
to as apoptosis (A0). Ao is typified by
deformation of the cell (blebbing/boiling membrane) membrane, cell
shrinkage, and condensation of the nuclear chromatin (due to endogenous
endonuclease activity on the genomic DNA).1,2 For the
majority of cells this is a constitutive response, but Ao
may also be induced in certain cells of the immune system by a variety
of stress mediators (ie, glucocorticoids, inflammatory cytokines such
as TNF, nitric oxide, etc) that are present during pathological
conditions including sepsis. In this respect, sepsis is reported to
induce an increase in the rate of Ao in immature
lymphocytes residing in hematopoietic tissues such as the thymus and
bone marrow.3,4 Alternatively, secondary lymphoid tissue
such as the spleen exhibit little innate (unstimulated) evidence of
Ao.
With respect to B lymphocytes, it was observed that cells of the bone
marrow exhibited evidence of increased B-cell Ao not observed in the splenic B cells.4 Whereas this may in part be explained by variation in the maturational state of these
lymphocytes, it may also reflect differences in local tissue exposure
to mediators and/or antigens released during polymicrobial
sepsis. Inasmuch, one would speculate that lymphoid cells resident in
the intestinal wall, such as Peyer's patch cells, would be exposed to
increased enteric antigenic burden because studies indicate that after
traumatic injury, shock, and/or sepsis, gut barrier
permeability is increased.5,6 Such exposure should lead
directly or indirectly to the stimulation of local mucosal lymphoid (T-
and/or B-cell) tissue, which contributes to local inflammatory
response. These mediators in turn may contribute to the suppression of
B- and T-cell responses that have been observed after trauma, shock,
and sepsis.7 The Peyer's patches are also the major site
in which mucosal B cells ultimately become committed to IgA
production.8 As the secretion of IgA is an important component in maintaining gut mucosal epithelia immune
function,9 and the production of IgA is an outcome of the
activation of the B cell,10 alterations in this response
may provide insight into the ongoing immune response developed in
response to a polymicrobial septic challenge. In this respect,
B-lymphocyte activation (as evidenced by nuclear factors
activation11,12 and subsequent IgA release) itself is
associated with increased Ao as a possible autoregulatory
mechanism with pathologic potential.13,14 We, therefore,
hypothesize that among the many factors that may play an important role
in the modulation of mucosal immunity during sepsis, is the induction
of Ao.
The aim of this study was to determine: (1) whether or not mucosal
lymphoid cells derived from the Peyer's patches show evidence of
increased Ao after polymicrobial sepsis; (2) if such
changes are detected, are they associated with alterations in mucosal lymphocyte function, such as IgA production and/or nuclear
factor activation; and (3) if such alterations are mediated by
endotoxin and/or Fas ligand-Fas antigen (Apo-1/CD95)
interaction.
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MATERIALS AND METHODS |
Cecal ligation and puncture (CLP).15
Male inbred C3H/HeN mice (endotoxin-sensitive)(Charles River, Portage
MI), C3H/HeJ (endotoxin-tolerant),16,17 or
C3H/HeJ-Faslgld mice (endotoxin-tolerant/Fas ligand
[FasL] deficient)18,19(Jackson Laboratory, Bar Harbor,
ME) 6 to 8 weeks of age were lightly anesthetized with metofane
[Methoxyflurane 2.2-Dichloro-1,1 difluoroethyl methyl ether. B.H.T.
(Butylated hydroxytoluene 0.01% wt/wt)]. A midline incision (1.5 to 2 cm) was made just caudal to the diaphragm, to expose the internal
organs. The cecum was isolated, ligated just below the ileocecal valve,
and punctured (CLP) in two places to induce sepsis. For the controls
(CLP-shams), the cecum was isolated but neither ligated or punctured.
The muscle layer and epidermal layer were sutured in layers and
xylocaine applied to the areas of the incision. Lactated Ringer's
Solution (0.6 mL) was administered subcutaneously in these and the sham animals.
The studies performed here were all carried out in accordance with the
National Institutes of Health Guidelines on Laboratory Animals and were
approved by the Rhode Island Hospital Committee on Animal Use and Care.
Peyer's patch lymphocyte isolation.
The Peyer's patches (4 to 6 patches per mouse) were excised
aseptically from the exposed small intestine of methoxyflurane-killed mice at 12 or 24 hours post-CLP or sham-CLP, then placed into petri
dishes (60 × 55 mL) containing 5 mL Hank's Balanced Salts Solution (HBSS). The Peyer's patches were gently glass ground, then
transferred into a 15 mL conical centrifuge tube, washed once with
Dulbecco's Modified Eagle's Medium (DMEM), and centrifuged at 400 × g for 10 to 15 minutes at room temperature. The pellet was disrupted, resuspended in 8 mL of DMEM, layered over 5 mL of 67%
Percoll, and centrifuged at 600 × g for 20 minutes. The cells at the interface were then harvested with a pipet and washed in
12 mL of DMEM.
Total viable cell yield.
Peyer's patch cell viability and total cell yield was determined by
trypan blue exclusion.
Cell staining and flow cytometric analysis.
In an attempt to correlate the changes in the percentage of cells that
were Ao+ with phenotypic expression, cells were stained with the combination of antibodies conjugated to either fluorescein isothiocyanate (FITC), phycoerythrin (PE) or Cy-Chrome,
and the DNA dye 4',6-diamino-2-phenylindole,
dihydrochloride (DAPI, Molecular Bioprobes Inc, Eugene, OR) for cell
cycle analysis according to the methods of Telford et al,20
as previously applied in this laboratory.4 In a typical
staining protocol, 2 x 106 cells were incubated with 10 µg nonspecific mouse IgG/mL phosphate-buffered saline (PBS),
containing 1.0% bovine serum albumin (BSA) and 0.1% sodium azide
(PBS-BSA-Az buffer), for 15 minutes at 4°C. The cells
were then washed by centrifugation and incubated 45 minutes
(4°C) with 25 µL of PBS-BSA-Az buffer containing 1 µg of monoclonal antibody against either the murine B-cell marker
CD45R [also known as B220 (clone RA3-6B2, rat IgG2a)], the mouse
Thelper-cell marker CD4 (clone RM4-5, rat IgG2a), or Fas
(Apo-1/CD95) (clone Jo2, hamster IgG) obtained from
Pharmingen Inc (San Diego, CA) conjugated to either FITC, PE, or
Cy-Chrome. This step was repeated for two- or three-color monoclonal
antibody staining. The cells were again washed as before and fixed for
no less then 30 minutes on ice in PBS containing 1% paraformaldehyde.
After fixation the cells were pelleted by centrifugation and the pellet
resuspended in DAPI staining reagent [0.1% Triton X-100, 0.1 mmol/L
EDTA disodium salt, 0.05 mg/mL RNase A (50 units/mg), 1 µg/mL DAPI,
in PBS pH 7.4] if the extent of Ao was to be determined.
Samples were stored in the dark at 4°C until analysis was carried
out (usually within 24 hours). Isotypic controls, ie, 1 µg of either
FITC-, PE-, or Cy-Chrome-conjugated rat IgG2a or FITC-conjugated
hamster IgG (Pharmingen Inc)/106 cells, were included so as
to allow us to assess the nonspecific antibody staining and gate this
out appropriately.
For multicolor analysis, DAPI was excited with a Coherenet ANOVA-70S
Spectrum laser (Spectrum Products Div, Palo Alto, CA) set to 360 nm and
fluorescent emission detected with a 424 ± 22 nm blue reflecting
dichroic filter. Alternatively, FITC, PE, and Cy-Chrome conjugates were
excited with an argon laser set at 488 nm, but detection of
FITC-fluorescent emission was with a 530 ± 15 nm wideband pass
filter, whereas PE emission was detected with a 575 ± 13 nm
dichroic filter and Cy-Chrome detected with a 670 nm long pass filter.
FITC, PE, Cy-Chrome, and/or DAPI emission overlap was corrected
by electronic compensation. FITC or PE single-positive as well as
double-positive and double-negative DAPI cell cycle analysis was
determined after gating of cell-debris and doublets for no less then
20,000 cells/sample. By using PC-Lysis Version 1.0 software (Becton Dickinson, San Jose, CA), FITC-, PE-, or Cy-Chrome-positive as opposed to fluorochrome-negative cells were established based on the fluorescent emission of the nonspecific FITC/PE isotypic antibody controls with two-color fluorescent cytograms. Histograms of the regionalized cells were then produced of
cell number versus DAPI stain intensity (DAPI fluorescent emission) from which the percentage of cells residing in various stages of the
cell cycle could be determined.
Alternatively, after the determination of B220+ versus
B220 negative regions, as described above,
histograms of the regionalized cells were then produced of cell number
versus anti-Fas-FITC stain intensity (mean channel fluorescence) from
which not only the percentage of cells that were Fas positive
(Fas+) could be determined based on the isotypic control
but also the intensity of Fas antigen expression (see Results section
for the results of typical two-color data for DAPI-cell cycle analysis and Fas antigen expression) could be determined.
Light and electron microscopic examination.
For histologic examination, Peyer's patches were excised and fixed
immediately by submersion in 4% buffered glutaraldehyde. After 1 to 3 hours of fixation, the buffered glutaraldehyde was then drawn off and
the tissues were rinsed with 0.1 mol/L phosphate buffer. The specimens
were then postfixed with osmium tetroxide for 1 hour, rinsed in
phosphate buffer, and stained en bloc with 2% uranyl acetate. Samples
were then dehydrated through a graded ethanol series followed by
propylene oxide. The tissue was then infiltrated overnight in a 1:1
mixture of propylene oxide and (Polybed-Araldite) epoxy
resin (Polysciences Inc, Warrington, PA). The following day, samples
are infiltrated for 8 more hours in Polybed-Araldite resin after which
they are embedded in fresh resin. Tissue blocks were polymerized for 2 days at approximately 74°C and then 1 µm sections were prepared
with an LKB Ultratome and stained with 1% toluidine blue for
examination by light microscopy. Areas selected for ultramicrotomy were
sectioned at 70 to 90 nm with a diamond knife and placed on 300-mesh
copper grids. Sections were stained with urannyl acetate for 1 hour and
lead citrate for 2 to 3 minutes. All sections were examined at 60 kV on
a Philips Model 301 transmission electron microscope (Philips Inc,
Mahwah, NJ).
IgA ELISpot assay.
The number of Peyer's patch cells secreting IgA was determined by an
Enzyme-linked Immunospot (ELISpot) assay.21 Cells were cultured (105/well) overnight on monoclonal antimouse IgA
(5 µg/mL; Biosource Inc, Camarillo, CA) precoated (overnight at
4°C), sterile Multiscreen-HA 96-well nitrocellulose filtration
plates (Millipore Corp, Bedford, MA). After washing and incubation
(overnight at 4°C) with a secondary biotinylated monoclonal
antimouse IgA (2.5 µg/mL; Pharmingen Inc, San Diego, CA), antibody
color was developed with
avidin-peroxidase-H202-ACE (3-amino-9-ethylcarbazole in 0.1 mol/L sodium acetate buffer [pH 5.0]). The number of spots present in the well was determined on a
Mocha Image analysis system (Jandel Scientific, Corte Madera, CA).
Assessment of B-cell nuclear expression of Rel(c-Rel and
RelB)/nuclear factor  B (NF-B) factors by western blot
analysis.
The nuclear appearance (expression) of Rel/NF-B factors, c-Rel, and
RelB, as an index of the potential nature of the in vivo B-cell
stimulant, was assessed by Western immunoblot analysis of the nuclear
extracts of B220+ cells harvested from the Peyer's patches of animals
24 hours after sham-CLP operation or CLP.
B220+ cells were selected (enriched) from 1 × 107
Peyer's patch cells, as previously described in our
laboratory,22 by binding to 4 × 107
antimouse B220 coated/goat antirat IgG magnetic M-450 Dynabeads (Dynal,
Inc, Great Neck, NY) per 5 mL and then gently stirring with a rotary
blood mixer (Robbins Scientific Corp, Sunnyvale, CA) for 30 minutes at
4°C. (4 × 107 antimouse B220 coated/goat antirat
IgG magnetic M-450 Dynabeads were created when approximately 1.5 × 108 Dynabeads in 10 mL of RPMI 1640 were coated
overnight at 4°C with monoclonal rat antimouse B220 [Pharmingen,
San Diego, CA] 10 µg/mL, and washed three times before use.) The
bead-bound cells (B220+) were then concentrated by using a
MCP-1 magnetic particle concentrator (Dynal, Inc) for 1 minute and the
unbound (B220 ) cells discarded. The cells that remained were
washed one more time, magnetically concentrated, and resuspended in
RPMI 1640 medium. Typical cell yield after positive magnetic selection
was approximately 4 × 106 cells total.
Nuclear fraction was then prepared by using a modification of the
methods of Sikora et al.23 Briefly, the nuclear fraction of
2 × 107 cells was prepared by the addition of two
volumes of 0.2% Nonidet P-40 in cell lysis buffer (10 mmol/L HEPES pH
7.9, 0.2 mmol/L EDTA, 1 mmol/L DDT, 0.5 mmol/L PMSF, and 10 mg
aprotinin/mL) to the washed cell pellet that was then incubated for 10 minutes on ice. The nuclei were pelleted by centrifugation at 3,300 × g for 15 minutes and the nuclear proteins extracted
from the pellet by resuspension in half the volume of extraction buffer
(20 mmol/L HEPES pH 7.9, 0.4 mol/L NaCl, 0.2 mmol/L EDTA, 1 mmol/L DDT,
0.5 mmol/L PMSF, and 10 mg aprotinin/mL), incubated for 30 minutes at
4°C, aliquoted, and stored at 70°C until required for
further assay.
The protein content of nuclear extract was determined fluorometrically
by using the NanoOrange Protein Kit (Molecular Bioprobes Inc, Eugene,
OR). The assays were carried out in a 96-well microculture dish
(Corning Scientific, Corning, NY) at a final reaction mixture volume of
250 µL and the change in fluorescence measured on a Bio-Tek FL 500 model, fluorescent plate reader (Bio-Tek Instruments, Winooski, VT).
Ten µg of nuclear extract protein (diluted in Tricine-gel sample
buffer) was separated on a precast 10% to 20% Tricine-sodium dodecyl
sulfate (SDS) polyacrylamide gel (Novex Experimental Technology, San
Diego, CA) electrophoretically24 by using a
Profile minigel electrophoresis system (Schleicher & Schuell, Keene,
NH) with EC 105 power supply (approximately 90 mA, 70 V for
approximately 1.5 hours; E-C Apparatus Corp, St. Petersburg, FL). The
separated proteins were then electroblotted (by using a Profile
miniblotter [Schleicher & Schuell, Keene, NH]; approximately 280 mA,
approximately 30 V, for approximately 1.5 hours) out of the gel onto
nitrocellulose (Novex Exp Tech). The blots were rinsed in Tris-buffered
saline containing 0.05% Tween-20 (TBS-Tween 20) and blocked for 2 to 4 hours with TBS-Tween 20 including 5% nonfat dry milk. Blots were then
washed 4 to 5 times (15 minutes per wash), incubated (4°C) with 3 to 4 mL of primary rabbit IgG polyclonal antibody directed against
either c-Rel or RelB (sc-070 and sc-226, respectively; Santa Cruz
Biotech, Santa Cruz, CA), or normal rabbit IgG control polyclonal
nonimmune sera (Santa Cruz Biotech) at concentration of 3µg/mL for 4 hours at 4°C in a 50 mL polyethylene centrifuge tube gently rotated
on a Robbins blood mixer (Robbins Sci Corp, Mountain View,
CA). After washing the blots 4 to 5 times, they were
incubated at room temperature for 1 hour with secondary polyclonal goat
antirabbit IgG antibody conjugated to alkaline phosphatase (1:1000;
Santa Cruz Biotech). Finally, after 4 to 5 washings the blots were
developed in alkaline phosphatase substrate buffer (100 mmol/L Tris,
100 mmol/L NaCl, 50 mmol/L MgCl2; pH 9.5) containing 330 µg nitroblue-tetrazolium and 168 µg 5-bromo-4-chloro-3-indolyl phosphate/mL for 45 minutes. Molecular weight (kD) of the proteins of
interest was determined by comparison with low molecular weight standards included in each gel. To quantify the differences present in
various nuclear extract samples, the band intensities were assessed
densitometrically by using a Mocha Image Analysis/SigmaGel work station
(Jandel Scientific, Corte Madera, CA).
Qualitative assessment of internucleosomal DNA fragmentation by gel
electrophoresis.
After washing, 3 x 106 total Peyer's patch cells were
extracted for genomic DNA by 10 minute lysis in 20 mmol/L Tris-HCl, pH 7.4, containing 10 mmol/L EDTA, and 0.2% Triton-X100. After removal of
cell debris the supernatant was incubated overnight with Proteinase K
(100 ug/mL, 50°C). The DNA was then chloroform/phenol extracted and
precipitated by using isopropanol (overnight, 20°C). After centrifugation, the pellet was dried in a SpeedVac Plus (Savant Instrument Inc, Farmingdale, NY) .
Genomic DNA was dissolved in 25 µL Tris-EDTA buffer containing 1 µg
RNase/mL and incubated for 1 hour (37°C). Five µL of 6 × TBE sample buffer was then added to each DNA sample, heated (65°C,
10 minutes) and then 15 µL applied to precast TBE 4% to 20%
polyacrylamide gel (Novex Experimental Technology). The samples and
standard (Lambda-DNA hindIII) were then electrophoresed for 90 minutes (90 to 100 V), stained with ethidium bromide, rinsed, visualized under UV light, and documented with Polaroid film (Polaroid Corp, Cambridge, CA).
Presentation of data and statistical analysis.
The data are presented as a mean and SE of the mean for each group.
Differences for total viable cell yields were considered to be
significant if P < .05, as determined by ANOVA (Spectrum Products Div) and a Tukey's test, was applied for multiple comparison. Differences in percentile data (ie, percentage of Ao+) were
considered to be significant if P < .05, as determined by
using the Mann-Whitney U test.
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RESULTS |
Polymicrobial sepsis induces a marked increase in the frequency of
Ao detected in Peyer's patches.
Figure 1A illustrates that a small but
statistically significant decline in total viable Peyer's patch cell
yield from endotoxin-sensitive C3H/HeN mice was detected at 24 hours
but not 12 hours after CLP. Alternatively, at 24 hours but not 12 hours, a significant increase in the frequency of cells that were
undergoing Ao was evident as detected by hypoploid DAPI DNA
staining ( Fig 1B). Evidence of endogenous endonuclease activity, as
detected by the fragmentation of genomic DNA, was also observed in the
DNA obtained from the septic-mouse Peyer's patch cells harvested at 24 hours post-CLP (Fig 2).
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| Fig 1.
Peyer's patch (A) total viable cell yield (as determined
by trypan blue exclusion) from C3H/HeN mice is significantly different 24 but not 12 hours after CLP. Sepsis (CLP) induces a significant increase in the percentage of mixed Peyer's patch cells that are determined to be (B) Ao+ by DAPI stain. Significance
indicated by * at P< .05 versus sham, Mann-Whitney
U test; Mean ± SEM; n = 6 mice sampled /group.
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| Fig 2.
Genomic DNA extracted from mixed Peyer's patch cells of
C3H/HeN mice typically exhibits increased evidence of endogenous
endonuclease activity in the form of DNA fragmentation as detected by
TBE-PAGE after the onset of sepsis (CLP).
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To determine whether the alterations observed in the ex vivo isolated
cells represented in vivo changes at the tissue level, we examined the
Peyer's patches harvested from C3H/HeN mice at both a light and
electron microscopic level for evidence of Ao. Figure 3A and C are representative samples
taken from a 24-hour postsham-operated mouse that exhibited typical
Peyer's patch morphology. However, Peyer's patches taken from mice 24 hours after onset of polymicrobial sepsis showed a marked change in
morphology (Fig 3B). With respect to the septic mouse Peyer's patches,
the majority of the apoptotic cells appear to be clustered in the
germinal center and not in the T-cell region localized more proximal to the mucosal epithelial layer. Closer inspection of these apoptotic clusters on an electronic micrographic level revealed karyorrhectic cells with condensed nuclei and what appear to be apoptotic bodies (Fig
3D).
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| Fig 3.
Marked changes in the C3H/HeN CLP mouse Peyer's patch
histology are evident at both the light microscopic (A v B)
level and at the electron microscopic level (C v D) 24 hours
postsham or CLP. (A) Illustrates typical lymphocyte morphology. The
figure is oriented with the mucosal epithelia (Mu) appearing on the
right side of the picture moving towards the germinal center on the left-hand side (magnification = 216X). (C) Shows normal lymphocytic morphology encountered at an electron microscopic level in the sham
mouse's Peyer's patch (magnification = 4825X). (B) Alternatively, shows changes typically encounter in the Peyer's patch of a septic mouse at a light microscopic level. Clusters of apoptotic cells (Apo),
with condense nuclei, can be observed, appearing to increase in
frequency within the germinal center of the CLP mouse's Peyer's patch, as opposed to the T-cell zone adjacent to the mucosal epithelia (Mu)(magnification = 216X). (D) Electron microscopic inspection of a
typically cluster of apoptotic cells within the germinal center showed
marked nuclear condensation (CN), cytoplasmic shrinkage, as well as
evidence of apoptotic fragmentation (Apo)(magnification = 4825X).
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Increased endogenous Ao detected in septic mouse Peyer's
patch cells is restricted to cells of the B- but not T-cell linage.
In an effort to delineate the population of C3H/HeN mouse Peyer's
patch cells undergoing Ao after the onset of sepsis, cells were concomitantly stained with the nonspecific murine B-cell marker
B220 (CD45) and the DNA dye DAPI.
Figure 4A through H illustrates the results
of typical two-color flow cytometric analyses of cell suspensions
obtained from the Peyer's patch 24 hours after sham-CLP (sham) or CLP
(simultaneously stained and analyzed). The contour plots in Fig 4A and
B show the typical cell cycle progression for sham or CLP-mouse
Peyer's patch cells and the primary region (R1) established around
these populations. In Fig 4C and D, the cell cycle histograms of cell number versus DNA content generated from the R1 region in Fig 4A and B
are shown. By using the sham, the histogram was divided into two
subregions (M1=apoptotic [Ao+] cell cycle region and M2=which includes G0/G1, S, and
G2/M cell cycle region, respectively). These regions were
kept consistent for all samples analyzed in a given experiment. With
respect to the sham, it can be observed that Peyer's patch cells
extracted from a septic mouse at 24 hours exhibited a population of
cells tailing off towards the lower left-hand corner with lower DNA
content from the G0/G1 peak (Fig 4A and B).
This is reflected by the increased percentage of apoptotic cells
detected in the M1 region of the histogram (Fig 4C and D). By using a
rat IgG-PE control, B220+ cells were discriminated from
B220 cells as depicted in Fig 4E and F and then cell cycle
histograms were produced (Fig 4G and H). Cell cycle histograms of DNA
content produced from each of these gated phenotypic populations
illustrate that the majority of the increase in apoptotic cells was
typically present in the B220+- stained cell population
(Fig 4G and H).
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| Fig 4.
(A through H) illustrates the results of typical
two-color flow cytometric analysis of a cell suspension obtained from
the Peyer's patch 24 hours after sham-CLP (sham)(A, C, E, G) or CLP (B, D, F, H; simultaneously stained and analyzed). (A and B) represents contour plots of the total cell sample delineated by DAPI fluorescence (FL4-A) versus cell size (width; FL4-W). The primary region (R1) typically established to enclose those cells in the various stages of
the cell cycle is also depicted. (C and D) are the cell cycle histograms of cell number versus DNA content (DAPI fluorescent intensity) generated from the R1 gated populations in (A and B). (E and
F) are the representative contour plots of these same cell samples
assessed by their phenotypic expression, ie, DAPI (FL4-A) versus B220
(FL2-H) fluorescent staining intensity. The nonspecific/negatively (-)
stained cells were delineated from the positively stained cells by the
use of isotypic antibody controls as indicated in the methods and these
regions are indicated as "B220-" or
"B220+" regions and the percentage of cells
expressing a given phenotype are given. Cell cycle histograms (G and H)
of cell number versus DNA content (DAPI fluorescent intensity) produced
from each of the phenotypically defined populations in (E and F),
respectively, illustrate the typical changes observed in frequency
apoptotic cells encountered after CLP in Peyer's patch cells.
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Figure 5 depicts the summary of the data
from no less than six independent animals. Peyer's patch samples were
examined in each group. Although a small decline in the percentage of
cells that are B220+ was observed in the CLP mice, this was
not statistically significant (Fig 5A). Alternatively, there was a
marked increase in the percentage of cells that were apoptotic in
septic mice, as compared with sham mice, as determined by cell cycle
analysis in the population of cells that were B220+ (Fig
5B). No similar change was evident in the B220 cell
population. Although not shown, combined staining with antibody against
the T-helper cell marker CD4 and DAPI did not show evidence of an increased frequency of Ao in this cell population
(data not shown).
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| Fig 5.
Phenotypic assessment of the percentage Ao+
cells from C3H/HeN mice indicates that marked augmentation of
Ao is present in the Peyer's patch cells is confined
primarily to B220+ (B lymphocytes) subpopulation but not
the B220- (B). This occurred despite the lack of
significant change in the frequency of B220+ cells (A).
Significance indicated by * at P < .05 versus sham, Mann-Whitney U test; mean ± SEM, n = 6 to 7 mice sampled
/group. Although not shown, Peyer's patch cells stained with antibody to CD4 (Thelper-cell) showed no change in the percentage
Ao+ cells.
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Polymicrobial sepsis induces an increase in the number of Peyer's
patch B cells that are secreting IgA.
To determine the potential functional significance of the increase in
B-lymphocyte Ao during sepsis, the ability of cells harvested from these animals to secret IgA was determined by using an
ELISpot method. The result in Fig 6
indicates that septic C3H/HeN mouse Peyer's patch cells exhibited a
marked increase in the number of cells secreting IgA.
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| Fig 6.
Sepsis (CLP) induces a significant increase in the number
of Peyer's patch cells from C3H/HeN mice secreting IgA as determined by ELISpot. Significance indicated by * at P < .05 versus
Sham, Mann-Whitney U test; Mean ± SEM; n = 5 to 6 mice
sampled /group.
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Polymicrobial sepsis induces an increase in the nuclear expression of
c-Rel but not RelB in Peyer's patch B cells.
Because recent studies11,12,25 indicate that mature B-cell
activation through the B-cell (Ig) receptor induces a marked increase
in the nuclear translocation/activation of the c-Rel component of the
Rel family of NF-B factors, as opposed to stimulants such as
endotoxin (ie, lipopolysaccharide) or CD40 ligand, which appear to
activate alternative components, such as RelB. We thought it should be
possible to obtain indirect insight as to the nature of the in vivo
B-lymphocyte stimulant acting on these cells after CLP. The results of
the western immunoblot analysis of the B220+ cell nuclear extracts
documented an increase in expression of c-Rel (approximately 75 kD) in
septic mice relative to sham (Fig 7A and
B). Alternatively, it was observed that RelB (approximately 68 kD) did
not appear to differ in its expression between sham and CLP mouse
samples. Although not shown, no staining in the molecular weight range
of either c-Rel or RelB with the control rabbit sera provided for these
antibodies was seen.
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| Fig 7.
CLP of C3H/HeN mice typically induced an increase in
B220+ cell nuclear extract c-Rel expression (A) as
compared to sham mouse cells at 24 hours observed by Western immunoblot
analysis. Alternatively, RelB expression (B) although evident is not
typically markedly different in extracts from sham or CLP mice. These
densitometric results are presented for three repeated independent
experiments.
|
|
The increased Ao detected in Peyer's patch cells during
sepsis is Fas-ligand but not an endotoxin-mediated process.
Because recent studies have indicated that lymphocyte activation is
associated with accelerated Ao that may be mediated via the
cell surface antigen known as Fas antigen and its associate ligand,26 we performed experiments to assess the expression of this antigen in the Peyer's patch cells at 12 and 24 hours after
the onset of polymicrobial sepsis.
Figure 8A through F illustrates the results
of typical two-color flow cytometric analyses of cell suspensions
obtained from the C3H/HeN mice Peyer's patch 24 hours after sham-CLP
(sham) or CLP concomitantly stained and analyzed for Fas antigen and B220. The histograms in Figure 8A and B show the typical Fas antigen staining pattern for sham- or CLP-mouse Peyer's patch cells over which
the hamster IgG-FITC control antibody staining was superimposed. By
using the isotype control the histograms were divided into two
subregions (M1 = Fas and M2 = Fas+).
These regions were kept consistent for all samples analyzed in a given
experiment. With respect to the sham, it can be observed that the mixed
Peyer's patch cells extracted from a septic mouse at 24 hours
exhibited a population of cells that are shifted to the right resulting
in an increase in the percentage of Fas+cells (Fig 8A and
B, as well as summary data for repeated experiments provided in
Fig 9A and B). By using a rat IgG-PE
control, B220+cells were again discriminated from
B220 cells as depicted in Fig 8C and D and then the
percentage of cells that were Fas+ was again determined as
depicted in the histograms in Fig 8E and F. These histograms of
Fas+ staining produced from each of the gated phenotypic
populations show that the majority of the increase in Fas+
cells was restricted to the B220+stained cell population
(Fig 8E and F, as well as summary data for repeated experiments
provided in Fig 10).
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| Fig 8.
(A through F) shows the results of typical two-color flow
cytometric analysis of a cell suspension obtained from C3H/HeN mice Peyer's Patches 24 hours after sham-CLP (sham)(A, C, E, G) or CLP (B,
D, F, H; simultaneously stained and analyzed). (A and B) are histograms
of cell number versus FITC-fluorescent intensity generated from the
ungated populations stain either antibody to Fas antigen (solid black
histogram) or isotypic antibody control (light gray overlayed
histogram)(summary data for repeated experiments is provided in Fig 9A
and B). The nonspecific/negatively (M1) stained cells were delineated
from the Fas-antigen positively (M2) stained cells by the use of
isotypic antibody controls. Similarly, B220+ cells were
discriminated from B220- cells by using isotypic Cychrome antibody control. The percentage of cells expressing a given phenotype are given (C and D). Histograms (E and F) of cell number v Fas antigen fluorescent intensity produced from each of the B220
phenotypically defined populations, respectively, illustrate the
typical changes observed in the frequency of Fas antigen positive cells
encountered after CLP in the Peyer's patch cells (summary data for
repeated experiments is provided in Fig 10).
|
|
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| Fig 9.
Assessment of the Fas antigen expression in mixed
Peyer's Patch cells from C3H/HeN mice illustrates that both the
percentage of cells that are Fas+ (A) as well as the Fas
antigen expression per cell (as shown by increased mean channel
fluorescence) (B) is increased at 24 hours after the onset of sepsis.
Signficance indicated by * at P< .05 versus sham,
Mann-Whitney U test; Mean ± SEM; n = 6 mice sampled
/group.
|
|
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| Fig 10.
Phenotypic assessment of the percentage
Fas+ cells indicates that marked augmentation of Fas
antigen expression present in the Peyer's patch cells from C3H/HeN
mice is confined primarily to the CLP mouse B220+ (B
lymphocytes) subpopulation but not the B220. This
occurred despite the lack of significant change in the Fas antigen mean
channel fluorescence of B220+ cells. Significance
indicated by * at P < .05 versus sham, Mann-Whitney U
test; mean ± SEM; n = 6 mice sampled/group. Although not shown, Peyer's patch cells stained with antibody to CD4
(Thelper-cell) showed no marked change in the percentage of
Fas+ cells or their mean channel fluorescence.
|
|
Summary data of no less that six animal's cells samples in each group
were presented in Fig 9A and B. At 12-hours post-CLP, no marked change
in Fas antigen expression was evident in the mixed Peyer's patch cell
population. However, by 24 hours a significant increase in the
percentage of cells that were positive for Fas antigen on septic mouse
cells was evident. Also although Fas antigen mean channel fluorescence
trended towards an increase in these septic mouse cells, it was not
statistically significant. Additionally, the percentage of Fas
antigen-positive cells was determined as a component of B220 antigen
expression. The results indicated that the increase in the percentage
of Fas antigen positive cells observed in the mixed septic mouse
Peyer's patch cells at 24 hours was primarily caused by a marked
increase in the percentage of cells that were B220+, which
were expressing Fas antigen and were not caused by changes in the
B220- population (Fig 10B).
In this respect, no increase in the percentage of Fas antigen positive
cells on cells expressing the T-cell phenotype, CD4 was observed (data
not shown).
A series of experiments were performed to determine if the increase in
Ao C3H/HeN mouse Peyer's patch B220+ cells was
due to either endotoxin (ETX; a product of gram-negative bacteria
observed in CLP mice) and/or the Fas ligand.18,19 Figure 11A illustrates that only in the
Peyer's patch cells harvested from endotoxin-tolerant C3H/HeJ
16,17 septic mice was a marked decline in viable cell yield
detected. Alternatively, although there was a slight decline in the
viable cell yield from the FasL-deficient
C3H/HeJ-Faslgld septic mice, it was not
statistically significant. Figure 11B shows that only the cells
obtained from CLP C3H/HeJ mouse and not the
C3H/HeJ-Faslgld mice exhibited increase in the
percentage of Ao+. This increase in the percentage of
Ao+ was also associated with a significant increase in the
percentage of Fas+ cells (Fig 11C). Here again, the enhancement in
percentage of Fas+ cells was restricted to the
B220+ cell population and was not a response to changes in
the B220 population
(Fig 12).
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| Fig 11.
Only Peyer's patch cells harvested from C3H/HeJ CLP
mice showed a marked decline in viable cell yield (A), which is
associated with a significant increase in both the frequency of
Ao (B) and percentage of Fas+ cells (C).
Significance indicate by * at P< .05 versus sham, Mann-Whitney U test; Mean ± SEM; n = 6 mice sampled
/group.
|
|
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| Fig 12.
Phenotypic assessment of the percentage of Fas+ cells
indicates that marked augmentation of Fas antigen expression is present in the Peyer's patch cells is confined primarily to the
B220+ (B-lymphocytes) subpopulation but not the
B220- of cells harvested from septic (CLP) C3H/HeJ mice.
Significance indicated by * at P< .05 v sham,
Mann-Whitney U test; mean ± SEM; n = 5 to 6 mice sampled/group.
|
|
| |
DISCUSSION |
The results presented here show that mucosal lymphocytes obtained from
Peyer's patches undergo a marked induction of Ao during sepsis. This is supported by evidence of augmented endonuclease activity in the septic animal's Peyer's patch cells (as detected by
both flow cytometry or TBE-PAGE, as well as by the increased presence
of morphologically apoptotic cells detected in the septic mouse
Peyer's patch germinal centers). The majority of these changes appear
to be restricted to cells of the B-cell lineage (B220+). It
could be argued that this increase in apoptotic frequency (from
approximately 3.5% in the sham to approximately 7.3% in CLP) might
not be sufficient to account for the approximately 2/3 reduction in
Peyer's patch viable cell yield. However, it should be noted that the
clearance of apoptotic cells is a dynamic process in which macrophages
are constantly involved in the removal of nonviable
cells.27 Hence, the accumulation of apoptotic cells at any
one time point in vivo would not necessarily have to be large. The
increase in Ao detected in these septic mice appears to be
associated with an augmented endogenous IgA secretory response. We
would speculate that this is in part a response to altered exposure to
enteric antigens released due to increased gut permeability observed
during sepsis.5,6 Inasmuch, this increase of Ao in Peyer's Patch B cells would appear to be an example of
activation-induced lymphocyte Ao.28,29
With respect to the kinetics of the apoptotic response exhibited here
in the Peyer's patch B220+ lymphoid cell population after
CLP, we observed that increased Ao was not evident until 24 hours and not at 12 hours after the onset of polymicrobial sepsis. A
similar observation was made with respect to viable cell yield.
Interestingly, although the delay in the expression of increased
Ao in the Peyer's patch cells differs from our previous
observations made with respect to cells of the thymus,3,4
in which evidence of an increased frequency of Ao was
detectable as early as 4 hours post-CLP; these findings are comparable
to our findings made with respect to bone marrow B-lymphocyte
Ao in which only late (24-hours post-CLP) after the onset
of sepsis was an increase in apoptotic frequency observed.4 However, these results differ from splenic B cells in which no detectable increase in innate Ao was observed up through
24-hours post-CLP. This supports the hypothesis that immune cells of a given linage, be they T cells,4 macrophages,30
and/or granulocytes,31 respond differently with
respect to the induction of apoptotic processes in response to
polymicrobial sepsis. This is due not only to differences in their
respective microenvironments, but their ontological/differentiation
status, as well as variation in the local concentration and/or
form of potential antigens and/or mediators capable of altering
the apoptotic process. In this respect, the increased apoptotic
response of mucosal B cells, such as Peyers patch cells examined here,
as compared to splenic B cells lack of Ao, we speculate is
a reflection of the difference in their exposure to enteric antigens
moving across the mucosal cell barrier. It has been well documented
that gut barrier function is reduced after trauma, shock,
and/or sepsis that would lead to a marked influx of such
enteric antigens.32,33 To the extent that the finding of
augmented B-cell Ao might be a peculiarity of Peyer's patches, we have recently observed in preliminary studies that B220+
lymphocyte subset of the lamina propria also exhibit a marked increase
in Ao during sepsis (24 hours; sham, 30.9 ± 2.3 v CLP; 40.0% ± 2.0%* Ao+, P< .05, n = 6/group). To the extent that this is a reflection of the teleological
linkage between which is thought to exist in the development of a
mucosal B-cell IgA-mediated response (antigenic stimulation of Peyer's
patch B cells [commitment to IgA production] leading to
differentiation, migration, through messenteric lymph nodes and
eventually to the lamina propria8) or a reflection of a
global nonspecific cellular intestinal response, must still be
determined. However, as we have not as yet assessed IgA production in
the Lamina propria B-cell population, it is unknown if this increased
Ao is comparably associated with activation of IgA release
in Peyer's patch cells.
The enhanced expression of Fas antigen is in line with the onset of
activation-induced B-cell Ao.29 However, the
mechanism underlying the induction of Fas antigen expression in this
cell population during sepsis remains unknown. FasL has been shown to
act as an inducer of its own receptor's expression19 in T lymphocytes whereby it acts to mediate lymphocyte activation-induced Ao.26,34,35 In this respect, we attempted to
determine the contribution of FasL-Fas antigen interaction to the
enhanced B220+ cell population Ao detected in
septic mouse Peyer's patch cells obtained from
C3H/HeN-FasLgld mice subjected to either sham-CLP
or CLP 24 hours earlier. This mouse strain contains a point mutation
that abolishes the capacity of FasL to bind Fas
receptor/antigen.19,36 By using FasL-deficient C3H/HeJ-Faslgld mice it was found that CLP did not
markedly enhance B-cell Ao in these animals' cells. These
findings indicate that FasL regulates not only the upregulation of Fas
antigen observed here but also the induction of Ao.
However, the source of FasL that might control this mucosal B-cell
process is less clear. B cells are not a typical source/producer of
FasL,35 most likely ruling out autocrine mediation of the
increased expression of Fas antigen in these cells. Alternatively,
paracrine release of FasL by Peyer's patch T cells and/or
nonlymphoid cells might mediate the increased expression of B-cell Fas
in septic mice. In this respect, although T cells are a documented
source of FasL,26,34,35,37 we did not detect increased Fas
antigen/receptor expression on the T-helper cell population. This indirectly suggests that T-cell FasL
expression/release is not markedly augmented in the Peyer's patch.
This, however, remains to established by direct measurement of either
cell surface FasL expression and/or its release.
Studies have also suggested that the stimulation of CD40 antigen on the
surface of the B cell during activation acts to upregulate the
expression of Fas antigen making the activated B cell more susceptible
to FasL-mediated apoptotic cell death.13,14 Here again the
B cell is not the primary source of CD40 ligand and the activated T
cell appears to provide this stimulant.13,14 However,
because the expression of CD40 antigen or CD40 ligand were not assessed
it is unknown whether either of these costimulatory components are
absent in the Peyer's patch cells from septic mice.
With respect to other mediators that might be implicated in the
induction of Ao in polymicrobial septic mouse lymphocytes, studies by Zhang et al38 reported that thymic
Ao could be induced by the exposure of mice to endotoxin, a
component of the cell wall of gram-negative bacteria (that is also a
polyclonal B-cell activator) commonly associated with systemic
infection associated with sepsis. However, the dosage of
lipopolysaccharide required to induce marked Ao was  50 µg/mouse (ip, 18 hours; equivalent to 2.5 mg/kg/body weight). This
dose of LPS typically produces blood levels of endotoxin that are
significantly higher then the circulating levels of this agent detected
in septic patients.39-41 It should also be noted that the
model of CLP used in this study induces the release of low levels of
circulating endotoxin (range 0.07 to 5 ng/mL serum) detectable as early
as 1 hour post-CLP, which steadily increases over the
first 24 hours thereafter to a maximum of approximately 60 ng/mL
serum.15,42,43 This model of sepsis is also polymicrobial
in nature as it is associated with not only gram-negative but also
gram-positive bacteria. Nonetheless, because studies have suggested the
role of endotoxin in the induction of thymic
Ao38,44 we examined the potential contribution
of endotoxin in increasing Ao in Peyer's patch lymphocytes
after sepsis.15,42,45,46 In this respect, the data we have
presented here by using the endotoxin-resistant C3H/HeJ mouse
strain16,17 show that the frequency of Ao is
not only increased in 24-hour CLP mice when compared with sham, but is
comparable to that observed in cells from septic endotoxin-sensitive
C3H/HeN mouse strain. These results would imply that endotoxin is not a
primary agent responsible for the induction of Ao in the
Peyer's patch. Furthermore, this also implies that the presence of
organisms not only of a gram-negative origin, but also gram-positive
bacteria46,47 and/or fungal agents may be required
to induce the enhanced Ao observed here.
The present results also show that polymicrobial sepsis and not the
stress associated with trauma, in the form of the midline laparotomy,
produces a marked increase in the frequency of Ao in
Peyer's patch lymphocytes.
The degree to which other inflammatory mediators released during
sepsis, such as cytokines like tumor necrosis factor, transforming growth factor- , lymphokines, prostanoids, steroids, etc, play a role
in the induction of mucosal B-cell Ao remains to be
examined.
With respect to the functional significance of the enhanced
Ao observed in the Peyer's patch B-lymphocytic population,
this appears to be a reflection of an antigen-specific response to the
influx of enteric antigens and not the response to nonspecific polyclonal mitogenic stimulation, such as might be mediated by endotoxin. This conclusion is based not only on our observation that
Ao is still enhanced in septic endotoxin-tolerant mouse
Peyer's patch cells but also on the ex vivo assessment of the nuclear factor footprint that is present in the cells obtained from the septic-mouse Peyer's patch. Neumann et al25 recently
reported that the activation of individual members of the nuclear
factor-B (NF-B) complex (eg, RelB and c-Rel) are differentially
affected (with respect to their translocation to the nucleus
and/or their transcription) by various B-cell stimuli. They
reported that the translocation and transcription of RelB was markedly
enhanced in cells that had been stimulated through the CD40 receptor as opposed to either the receptor for endotoxin (ie, lipopolysaccharide) or the Ig receptor of the B-cell receptor complex (which is evident but
weak). Alternatively, stimulation through the Ig receptor of the B-cell
receptor complex induced marked translocation of c-Rel to the nucleus,
but only a weak activation/translocation when stimulated through the
CD40 antigen and no marked translocation of c-Rel was observed with
endotoxin. Such a footprint of NF-B nuclear
translocation48 may provide some insight into the nature of
the stimulant responsible for the induction of IgA secretion in the
septic mouse Peyer's patch cells and the associated increase in
activation-induced Ao observed here. The results of the
western immunoblot analysis of nuclear protein extracts obtained from these septic animals supports the suggestion that the B-cell activation observed here is most likely Ig receptor-mediated, hence a specific response to enteric antigens, and does not appear to be due to nonspecific polyclonal B-cell activation by agents like endotoxin. However, although we speculate here that such a relationship exists, direct linkage between increased B-cell IgA release, increased c-Rel
translocation, and the eventual induction of Ao remains to
be determined.
In summary, our findings indicate that mucosal B-cell Ao
appears to be markedly increased by polymicrobial sepsis and that this
appears to be the result of a FasL-mediated process. Evidence of
antigen-specific polyclonal activation suggests the possible induction
of B-cell Ao and eventual germinal center B-cell functional impairment.
| |
FOOTNOTES |
Submitted May 12, 1997;
accepted October 6, 1997.
Supported by grant No. R01-GM53209 from the National Institutes of
Health, Bethesda, MD.
Address correspondence to Alfred Ayala, PhD, Center for Surgical
Research, 211 Middle House, Rhode Island Hospital, 593 Eddy Street,
Providence, RI 02903.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr Louis King at Michigan State University as well as Sally A. Johnson and Paul Monfils at the Central Research facilities at Rhode
Island Hospital for their assistance with the flow cytometry.
| |
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Abstract
Introduction
Abstract