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Division of Nephrology, Department of Medicine, Tupper Research Institute, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts
Correspondence: Bertrand L. Jaber, M.D., Division of Nephrology, New England Medical Center, 750 Washington St., Box 391, Boston, MA 02111. E-mail: bjaber{at}lifespan.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: polymorphonuclear cells • programmed cell death • chronic renal failure • toxins • dialysis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Interest in apoptosis has expanded with the recognition that enhancement of this constitutive cell death process in immune competent cells may contribute to the impaired host defense characteristic of many disease processes. Professional human phagocytes, including neutrophils and monocytes, undergo cell death via apoptosis when maintained in vitro. Mature human neutrophils undergo spontaneous apoptosis most rapidly, resulting in the demise of >50% within 48 h [8 ].
Although there is evidence that in uremic patients, mononuclear cells such as T lymphocytes and monocytes undergo accelerated apoptosis [9 10 11 12 ], little is known of neutrophil apoptosis in uremia and the factors that influence it. We recently observed that neutrophils from uremic patients undergo accelerated apoptosis [4 ] and that uremic plasma is apoptogenic [13 ]. Neutrophils undergoing apoptosis exhibit a dysfunctional pattern that is similar to that seen in uremic cells [4 14 ], suggesting that apoptotic cell death may be biologically relevant in uremia by contributing to cellular malfunction. However the triggering factor(s), mechanisms, and consequences of neutrophil apoptosis in uremia have not been adequately investigated.
The objectives of this study were to examine (1) the constitutive cell surface coexpression of Fas (also known as APO-1 or CD95) and FasL by neutrophils isolated from healthy subjects and patients with chronic renal failure (CRF) and end-stage renal disease (ESRD); (2) the susceptibility of uremic neutrophils to Fas-mediated apoptosis; and (3) whether uremic soluble factors can influence Fas and FasL expression by neutrophils.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of neutrophils
Water, cell culture media, and other solutions used in the study
were subjected to ultrafiltration using a polyamide hollow-fiber
ultrafilter (U2000; Gambro AB, Hechingen, Germany), to remove
cytokine-inducing agents. Neutrophils were harvested by Percoll
gradient (63–69%) followed by hypotonic erythrocyte lysis as
previously described [15
]. In brief, 5 mL of 63%
Percoll were underlayered with 5 mL of 69% Percoll (Sigma Chemical
Co., St. Louis, MO). Each 5-mL sample of blood was overlayered on the
63–69% gradient and centrifuged at 450 g for 25 min at
room temperature.
After harvesting the neutrophil cell layer, cells were washed in Dulbecco’s phosphate-buffered saline (PBS) (Gibco-BRL Life Technologies, Grand Island, NY) centrifuged at 450 g for 5 min at 4°C. Residual erythrocytes were subjected to hypotonic lysis. Cells were suspended in ice-cold, calcium-free PBS, and neutrophils were counted using a standard hemocytometer. The purity of neutrophils was <95%, and viability was <99% as judged by the trypan blue exclusion method. For immune staining conditions, neutrophils were suspended at 20 x 106/mL in PBS (without calcium and magnesium) + 5 mM Na2-EDTA, and for culture conditions, neutrophils were suspended in RPMI-1640 (pH 7.4; Sigma) supplemented with 10 mmol/L of L-glutamine, 24 mmol/L of NaHCO3 (Mallinckrodt, Paris, KY), 10 mmol/L of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Sigma), 100 U/mL of penicillin, 100 µg/mL of streptomycin (Irvine Scientific, Santa Ana, CA), and 10% autologous serum or fetal calf serum.
Detection of apoptosis-related molecules by flow cytometry
Cell surface expression of Fas
On isolation from each of the subjects, cell surface expression
of Fas was assessed by direct-immunofluorescence flow cytometry. In
brief, 50 µL of neutrophil suspension (106 cells) were
incubated with 50 µL of antihuman Fas monoclonal antibody (mAb)
UB2-fluorescein isothiocyanate (FITC) (Beckman Coulter, Miami, FL) or
irrelevant murine immunoglobulin (Ig) G1-FITC (Beckman Coulter) for 60
min in the dark at 4°C. Cells were washed once with PBS and fixed in
1% paraformaldehyde in PBS and stored in the dark at 4°C before
analysis. The mean channel of fluorescence intensity (MCF) was
calculated by subtracting the MCF of the appropriate negative control.
Cell surface expression of FasL
Cell surface expression of FasL was assessed by
indirect-immunofluorescence flow cytometry. In brief, 50 µL of
neutrophil suspension (106 cells) were first incubated with
20 µL of matrix metalloproteinase (MMP) inhibitor KB 8301 (20 µM)
(Pharmingen, San Diego, CA) to minimize processing and release of
membrane-bound FasL into its soluble form. For primary staining, 100
µL of biotinylated murine antihuman FasL mAb NOK-1 (
2 µg)
(Pharmingen) or irrelevant biotinylated murine IgG1
(Pharmingen) were then added to the cells and allowed to incubate for
an additional 60 min at room temperature in the dark. Cells were washed
one time with PBS containing 1% fetal bovine serum and 0.1% sodium
azide. After the supernatant was discarded, 100 µL (2 µg) of
streptavidin-phycoerythrin (Pharmingen) were added to the cell pellet,
and the mixture was incubated for 20–30 min at room temperature in the
dark. After secondary staining, cells were then washed one time with
PBS containing 1% fetal bovine serum and 0.1% sodium azide and were
fixed in 1% paraformaldehyde in PBS and stored in the dark at 4°C
before analysis. MCF intensity was calculated as previously described.
Neutrophil culture conditions
Neutrophils (2.5x106) were incubated in RPMI 1640
supplemented with 10% autologous serum with or without 200 ng/mL of
proapoptotic anti-Fas IgM mAb CH-11 (Beckman Coulter). After 24 h
of incubation at 37°C with 5% CO2, aliquots of
neutrophils were processed for quantification of apoptosis.
Additional experiments were performed to examine the influence of uremic soluble factors on Fas and FasL expression by neutrophils. In brief, neutrophils (20x106 cells) harvested from healthy subjects were incubated in RPMI 1640 supplemented with blood type ABO-compatible 10% normal or uremic serum. After the incubation period, total cell-associated Fas and FasL protein expression was measured by Western blot analysis.
Apoptosis measurement by the Annexin V-FITC staining method
Annexin V is a phospholipid-binding protein that has a high
affinity for phosphatidylserine, which is externalized on the cell
membrane during early stages of apoptosis. Based on these morphological
features, immunofluorescence of Annexin V-FITC (Coulter Corp., Opa
Locka, FL) binding to phosphatidylserine was used as a sensitive early
measure of neutrophil apoptosis. In brief, neutrophil suspensions
(0.5x106 cells) were washed one time with ice-cold PBS,
and the pellet was resuspended in 490 µL of ice-cold binding buffer.
Annexin V-FITC and propidium iodide (PI) were added, and the mixture
was incubated on ice for 10 min in the dark at room temperature. PI was
used to recognize late apoptotic and necrotic cells. Cells were then
immediately analyzed by flow cytometry or fixed in 1% paraformaldehyde
in PBS and stored in the dark at 4°C before analysis. Cells that
stained only for Annexin V were considered apoptotic, and cells that
dually stained for both Annexin V and PI were considered to have
undergone secondary necrosis.
Flow-cytometric analyses
All flow-cytometric analyses was carried out at a flow rate of
1,000 events/s using a dual-laser flow cytometer (EPICS® XL-MCL,
Coulter Corp., Miami, FL). A total of 10,000 events were counted. Cell
debris and clumps were excluded from the analysis by gating single
cells in the forward and side light scatters. FITC, PI, and
phycoerythrin were excited using the 488-nm UV line of the argon laser.
The data were analyzed with personal-computer-based software
(WinMDI version 2.8; Scripps Research Institute, La Jolla,
CA).
Neutrophil-associated Fas and FasL identification by immunoblot
After 24 h of incubation, neutrophil suspensions were
centrifuged at 450 g for 5 min at room temperature, and
supernatant was discarded. The cell pellets (50 x 106
cells) were lysed in sample buffer (10 mM Tris-HCl, pH 7.6, 140 mM
NaCl, 1% Triton X-100, 5 mM EDTA, and 2 mM phenylmethylsulfonyl
fluoride) and incubated at 4°C for 30 min. The protein concentration
was determined using the Bradford reagent (Sigma). Proteins were
denatured by boiling in sample buffer [0.25 M Tris-HCl, pH 6.8, 10%
glycerol, 2% sodium dodecyl sulfate (SDS), 5% ß-mercaptoethanol,
and 0.1% bromophenol blue] for 5 min. The samples were
electrophoresed in a 10% SDS-polyacrylamide gel and then transferred
to a nitrocellulose membrane. The membranes were blocked in 5%
blocking reagent for an hour, washed in PBS–0.1% Tween 20, and
incubated with mouse anti-human Fas mAb (Oncogene Research Products,
Cambridge, MA) or rabbit anti-human FasL polyclonal antibody (Oncogene)
overnight at 4°C. After the incubation period, samples were washed in
PBS–0.1% Tween 20 and incubated with horseradish
peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody for 60 min
at room temperature. Immunoblots were developed using an enhanced
chemiluminescence protein detection kit (Amersham, Arlington Heights,
IL). Densitometric analysis of the images was performed using the Scion
Image ß3b acquisition and analysis software (Scion Corp., Frederick,
MD). Optical density (OD) calibration was used for all analyses, using
a generic mapping of brightness to OD. This calibration was used for
comparison of lane bands in the same digital image.
Statistical analysis
Statistical analyses were performed using the InStat software
package version 2.01 (Graph Pad, San Diego, CA). Comparisons between
groups were made by ranked nonparametric Kruskal-Wallis analysis of
variance and two-tailed Mann-Whitney tests (unpaired and paired) for
continuous variables. All results were expressed as percentages or
means plus or minus standard errors. Spearman rank correlation
coefficients were used to test for association between Fas/FasL
expression, apoptosis and creatinine clearance as measured by the
Cockcroft and Gault equation. Differences were considered statistically
significant at two-tailed P values of 
0.05.
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RESULTS |
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Among control subjects and patients with CRF, there was an inverse correlation between creatinine clearance and unstimulated (r=-0.413; P=0.04) (Fig. 4 ) as well as Fas-stimulated (r=-0.572; P=0.004) apoptosis (Fig. 4) . Furthermore, there was a positive correlation between unstimulated and Fas-stimulated neutrophil apoptosis (r=0.746; P<0.0001) and between cell surface Fas expression and Fas-stimulated apoptosis (r=0.347; P=0.03) (Fig. 5 ). There was no correlation between age and apoptosis (P=0.543) or age and Fas (P=0.34) or FasL (P=0.96) expression. Finally, among patients with CRF, there was an inverse correlation between the circulating white blood cell count and neutrophil apoptosis (r=-0.611; P=0.035).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The biological importance of the Fas/FasL system has been studied extensively in T lymphocytes. Indeed, activated cytotoxic T cells can deploy FasL as death effector molecules in their strategies to induce killing of Fas-bearing target cells [20 , 21 ]. In recent years, studies have shown that mature human neutrophils coexpress constitutively both Fas and FasL [22 , 23 ]. Dual expression of these apoptosis-modulating proteins is in agreement with the natural in vitro and in vivo fate of these cells. In fact, neutrophils undergo PCD when maintained in vitro [22 23 24 ]. In addition, in vivo, circulating blood neutrophils have a half-life of approximately 6 h, after which they migrate into tissues where they undergo PCD [25 ] and are recognized and engulfed by tissue-derived macrophages [26 ].
The objectives of the present study were to evaluate whether the constitutive coexpression of Fas and FasL proteins by neutrophils is increased among patients with various degrees of CRF and whether these cells were more sensitive to a Fas-mediated death track. Our findings indicate that neutrophils from patients with CRF, unlike normal neutrophils, undergo increased in vitro apoptosis. This is paralleled by increased cell surface expression of both Fas and FasL and a relatively higher susceptibility to Fas-mediated apoptosis with progressive renal failure. In addition, uremic serum up-regulates Fas and FasL expression by normal neutrophils, suggesting an inducing role by uremic retention solutes.
Our results are in agreement with a recent report demonstrating an
inverse correlation between creatinine clearance and apoptosis of
peripheral blood mononuclear cells among patients with CRF
[12
]. However, our observations do not support the
results of our previous studies demonstrating that neutrophils from
patients on maintenance HD are more apoptotic than cells from healthy
individuals [4
]. Differences in neutrophil separation
techniques, type of culture medium, apoptosis detection assays, patient
characteristics, and sample size may account for the different results.
Furthermore, since neutrophil apoptosis is inhibited in vitro after
treatment with bacterial lipopolysaccharide, complement component C5a,
TNF-
, interleukin-1ß, or granulocyte-macrophage colony-stimulating
factor [27
28
], we cannot exclude the possibility that
the prolonged cell survival of neutrophils from HD patients reported
here was due to recurrent in vivo exposure to dialysate bacterial
contaminants and/or proinflammatory cytokines.
Fas expression by neutrophils was increased in undialyzed uremic patients and was paralleled by higher apoptosis. Matsumoto et al. [9 ] observed similar findings in T lymphocytes, arguing that the lymphopenia commonly observed in patients with CRF may be due in part to increased apoptosis. These replicated data in two different subsets of leukocytes suggest a common cascade of events leading to the increased in vitro demise of leukocytes in uremic patients. In support of this hypothesis, we observed an inverse correlation between white blood cell counts and neutrophil apoptosis among patients with CRF.
Although FasL is expressed predominantly on the surface of activated T
and B cells [29
, 30
], FasL-bearing T cells
can produce soluble FasL (sFasL) effector molecules, which result from
cell surface cleavage of the membrane-bound form of FasL (mFasL). Since
processing of TNF- precursors is dependent on at least one MMP
[31
], studies have indicated that FasL cell surface
processing also requires the action of MMP [32
]. Since
neutrophil-associated oxidative metabolism can activate MMP
[33
], we used in our experiments MMP inhibitor KB 8301
[32
] to stabilize the mFasL moiety and allow its cell
surface detection by flow cytometry. We propose that coexpression of
both Fas and FasL by neutrophils from patients with CRF may act in
concert, whereby apoptosis is mediated by interaction of Fas and mFasL
as well as sFasL, in an autocrine and paracrine way, respectively.
We did not fully explore the mechanisms for increased Fas and FasL expression by neutrophils in CRF. However, we found that, although uremic serum increased cell-associated synthesis of both proteins, urea alone, a surrogate marker of small-molecular-weight uremic toxins, had no impact on Fas expression (data not shown). We have previously reported on the apoptogenic potential of uremic serum [4 , 13 ] and more recently have observed detectable levels of both sFas and sFasL in serum of patients with renal failure [34 ]. These findings suggest a complex interplay of autocrine, paracrine, and endocrine Fas-FasL interactions as a mechanism of cellular apoptosis in uremia.
The detection of FasL on the cell surface of neutrophils has been the subject of ongoing debate. Indeed, whereas some authors have argued that mature human neutrophils coexpress constitutively both Fas and FasL [18 , 22 , 23 ], more recent studies failed to demonstrate FasL expression by peripheral blood neutrophils [35 ]. These studies were performed with neutrophils harvested from healthy volunteers and therefore do not in any way reflect on disease states. The detection of FasL by neutrophils in uremic patients is in agreement with recent studies by Ranjan et al. [36 ], demonstrating that uremia also results in increased FasL expression by mononuclear cells [36 ].
Although the execution of Fas-mediated apoptosis is largely independent of reactive oxygen intermediates (ROIs), both Fas and FasL expression can be induced by ROIs [37 ]. Studies by Ward et al. (38) and Klein et al. (39) have shown that uremic serum primes neutrophils to higher basal ROI production, mainly hydrogen peroxide. Consequently, uremic soluble factors via ROI generation, may induce in part the Fas and FasL expression by neutrophils, resulting in their accelerated demise.
In summary, our findings indicate that the susceptibility of neutrophils to spontaneous and Fas-mediated apoptosis differs between cells from healthy and uremic individuals. This susceptibility is determined in part by the level of renal function, indicating that uremic retention solutes may result in cellular derangement that promotes PCD. Further studies are under way to examine factors involved in the transcriptional regulation of Fas and FasL expression in uremia, as well as the role of ROI in the activation of the Fas/FasL pathway.
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ACKNOWLEDGEMENTS |
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This work was presented in part at the 32nd Annual Meeting of the American Society of Nephrology, Miami, FL, November 5–8, 1999.
Received June 28, 2000; revised December 10, 2000; accepted December 12, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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processing by a metalloproteinase inhibitor Nature 370,558-561[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and black (•) circles represent unstimulated and
Fas-stimulated apoptosis, respectively. The broken (–) and solid
(––) lines represent the correlation slopes between creatinine
clearance and unstimulated apoptosis (r=-0.413;
P=0.04) or Fas-stimulated apoptosis (r=-0.572;
P=0.004), respectively.
