Immunogenetics Laboratory and
Flow Cytometry Unit, National Institute for Cancer Research, Genoa, Italy; and
* Department of Internal Medicine, University of Genoa, Genoa, Italy
Correspondence: Guido Frumento, M.D., Immunogenetics Laboratory, National Cancer Institute c/o Advanced Biotechnology Center A2, Largo Rosanna Benzi 10, 16132 Genova, Italy. E-mail: frumento{at}ermes.cba.unige.it
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Key Words: human • neutrophils • age-related • apoptosis
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RIII (Coulter-Immunotech, Westbrook, MA), and anti-human
CD32/FcRII (Coulter-Immunotech). The mouse anti-human CD8 Leu-2b mAb
(Becton Dickinson, San Jose, CA) was used in some tests as an
irrelevant first antibody. The secondary antibodies, anti-mouse
IgG1/PE (phycoerythrin) and anti-IgG2a/PE, were purchased from
Southern Biotechnology (Birmingham, AL), and the anti-mouse IgG +
IgM/FITC was from Pierce (Rockford, IL). All antibodies were titrated
before use. To obtain the F(ab')2 fragments, the W6/32 IgG was digested by pepsin. Briefly, the antibody was incubated overnight at a concentration of 1 mg/mL in 100 mM citrate buffer, pH 3.5, in the presence of 700 µg/mL pepsin (Calbiochem-Novabiochem, San Diego, CA). Afterward, neutrality was restored by adding one-tenth the volume of Tris-HCl buffer (pH 8.8), the solution was passed through a Protein A Sepharose CL-4B column six times and centrifuged in Centricon 30 concentrators (Amicon, Beverly, MA), and the high molecular-weight fraction was recovered and dialyzed in PBS. An aliquot of the sample was run in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) without finding undigested IgGs. The F(ab')2 fragments of the anti-CD16 3G8 mAb were purchased from Medarex (Annandale, NJ).
Human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased from Genzyme Co. (Cambridge, MA). The Annexin V/FITC kit was purchased from Boehringer Ingelheim (Heidelberg, Germany). Propidium iodide, dibutyryl adenosine 3',5'-cyclic phosphate (cAMP), and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma. Proteinase K and RNAse A were from Boehringer Mannheim.
Cell isolation and culture
Heparinized (heparin 10 units/mL) venous blood was obtained from
healthy male volunteers after informed consent. Anticoagulated blood (5
mL) was layered on a discontinuous density gradient of Ficoll-Hypaque
(5 mL) over Mono-Poly Resolving Medium (3 mL) in 15 mL conical-bottom
polystyrene tubes (Falcon, Becton Dickinson, Franklin Lakes, NJ). The
tubes were centrifuged at 500 g for 60 min. Thereafter, the
neutrophil fraction was recovered and layered on 1 mL Mono-Poly
Resolving Medium for subsequent centrifugation (30 min). The resulting
neutrophils were washed three times and checked for purity by May
Grunwald-Giemsa staining and viability by ethidium bromide-fluorescein
diacetate test.
Cells were then resuspended in the above-mentioned medium at a concentration of 2 x 106/mL, and 0.5 mL of the cell suspension was incubated in tissue culture tubes (17x100 mm, Falcon) at 37°C in a 5% CO2 atmosphere for the length of time indicated in the text. Some experiments were carried out in the presence of GM-CSF (final concentration, 10 ng/mL), cAMP (1 mM), or PMA (1 and 5 ng/mL).
The effect of Class I ligation was tested by culturing freshly isolated neutrophils for 24 h in culture medium supplemented with 20 µg/mL solution of W6/32 F(ab')2 fragments or 3G8 F(ab')2 fragments. PBS (50 µL) was added to the control samples.
Flow cytometric analysis
For direct immunofluorescence, 2 x 107 cells
were incubated for 30 min at 4°C with the appropriate concentration
of FITC-conjugated W6/32 in ice-cold PBS plus 5% mouse serum, washed
twice, and resuspended for cell sorting. The medium used for the other
samples was ice-cold PBS supplemented with 3% FCS and 0.1% sodium
azide. For indirect immunofluorescence, 5 x 105 cells
were washed, and the pellet was incubated for 30 min at 4°C with
appropriate concentrations of primary antibody, washed twice, incubated
for 30 min at 4°C with the appropriate concentration of the secondary
antibody, washed twice, and resuspended for flow cytometric analysis.
When indicated, cells were incubated with an irrelevant first antibody
followed by the above-mentioned secondary antibody from Pierce. For
dual labeling of CD16 and Class I, neutrophils were incubated with 3G8,
washed, incubated with anti-mouse IgG1/PE, washed, incubated with
FITC-conjugated W6/32, washed again, and resuspended for flow
cytometric analysis. For dual labeling with use of Annexin V/FITC, an
indirect immunofluorescence with W6/32 followed by anti-mouse IgG2a/PE
was initially performed. Then Annexin V/FITC was added with minor
changes to the manufacturer’s instructions. Briefly, cells were
resuspended in 100 µl isotonic binding buffer, and 3 µl Annexin
V/FITC was added. After 15 min, cells were washed and resuspended in
the buffer for flow cytometric analysis.
The method of Nicoletti et al. [18 ] was used, with minor changes, to evaluate propidium iodide incorporation. Briefly, cells were washed and resuspended in 0.5 mL PBS, the cell suspension was added dropwise to 4.5 mL ice-cold 80% ethanol while vortexing, and kept at -20°C for 24 h. Afterward, cells were washed twice, propidium iodide was added to a final concentration of 10 µg/mL, and the sample was analyzed by flow cytometry after incubation overnight. Flow cytometry analysis was performed on an EPICS XL flow cytometer (Coulter, Hialeah, FL). Briefly, living granulocytes were gated on the basis of physical properties (forward vs. side-light scatter), and at least 2000 living cells were analyzed per sample.
Cell sorting
For cell sorting, neutrophils were cultured for 24 h to
achieve a bimodal distribution of HLA Class I molecule expression.
Cells were then labeled with FITC-conjugated W6/32 to detect
granulocytes with normal expression of HLA Class I and those with low
HLA Class I expression. Cell sorting was performed on an EPICS Elite
cell sorter (Coulter), following identification of the correct drop
delay using the "dot matrix" procedure. A mean frequency of
1500–2500 cells/sec was used, and the coincident granulocytes were
excluded. During and after sorting, cells were kept at 4°C. The
purity of the two sorted populations (either high HLA or low HLA Class
I-expressing granulocytes) was immediately checked on the
above-mentioned flow cytometer. The sorted subpopulations were further
used for the evaluation of propidium iodide incorporation, DNA
fragmentation, and cell morphology.
DNA electrophoresis
Neutrophils (2x106) from the two sorted populations
were washed twice in PBS. Pellets were incubated overnight at 37°C in
0.5 mL lysis buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0, 200 µg/mL
proteinase K, 4% SDS, 200 mM NaCl). After adding 15 µl of 10 mg/mL
heat-treated RNAse A, each sample was incubated for 1 h at 37°C.
After addition of 15 µl of 10 mg/mL proteinase K and another 1 h
incubation at 37°C, samples were treated twice with 1 vol
phenol:chloroform:isoamyl alcohol (25:24:1) and once with 1 vol
chloroform:isoamyl alcohol (24:1). After overnight precipitation with
0.1 vol 5 M NaCl and 2 vol ethanol at -20°C, pellets were air-dried
and resuspended in TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA).
DNA concentration was then evaluated by spectrophotometric (260 nm)
analysis, samples (5 µg DNA/lane) were added to loading buffer (2.5%
Ficoll, 0.04% bromophenol blue, 0.04% xylene cyanole), heated at
60°C for 10 min, and separated at 10 V in 1.5% agarose gel
containing 1% ethidium bromide.
Light-microscopy assessment of neutrophil apoptosis
Cells from the two sorted populations were cytocentrifuged,
fixed, and stained with May-Grünwald-Giemsa. Thereafter, slides
were read blindly by two independent observers using oil-immersion
light microscopic examination of at least 500 cells/slide (1000x
magnification). Cells showing apoptotic morphology were identified,
according to the following criteria: cell shrinkage, nuclear
condensation and fragmentation, and plasma membrane ruffling and
blebbing.
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97%, and viability was >98% at the beginning of the
culture. The possibility that necrotic cells or cellular debris could
account for Class I downregulation was ruled out also by gating and
analyzing only cells having the physical properties (in terms of size
and lateral light-scatter) of living neutrophils.
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Figure 1. Time course of Class I expression in aging neutrophils. Indirect
immunofluorescence was performed using the anti-HLA Class I MoAb W6/32
on freshly isolated neutrophils (A) and after 8 h (B), 24 h
(C), and 36 h (D), in culture. Dotted curves show fluorescent
intensity of indirect immunofluorescence with an irrelevant first MoAb.
A representative experiment of the two performed is shown. After
24 h in culture, 51% of neutrophils expressed low levels of Class
I in this experiment, and 69% did so after 36 h in culture.
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The hypothesis that the modulation of Class I expression could be related to neutrophil activation during in vitro culture was verified also. In fact, activation with PMA at 1 and 5 ng/mL did not alter the expression of Class I molecules on cell membranes (unpublished results). Thus, our results suggest that separation of cultured neutrophils into two subpopulations, one with normal and one with reduced Class I expression, is an apparently spontaneous phenomenon.
Downregulation of Class I is related to downregulation of CD16
To evaluate the selectivity of the modulation of HLA Class I
expression, together with Class I, a panel of surface molecules
expressed on granulocytes was studied by indirect immunofluorescence.
These included CD16, downregulated in apoptotic neutrophils
[16
, 17
]; CD32, which does not change
[16
]; and CD15. The modulation of CD15 has never been
investigated previously. At the beginning of culture, the four proteins
were characterized by a unimodal distribution (unpublished results).
Although the expression levels of CD15 and CD32 remained unchanged
during culture, granulocytes analyzed for the expression of Class I and
CD16 split into two subpopulations of normal or low-expressing cells
(Fig. 2
).
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Figure 2. Effect of culturing on membrane antigen expression in human
neutrophils. After 24 h in culture, neutrophils were tested by
indirect immunofluorescence using an anti-Class I (A), anti-CD15 (B),
anti-CD16 (C), and anti-CD32 (D) MoAb, respectively, as the first
antibody. A representative experiment of the two performed is shown.
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Figure 3. Coincidence of Class I and CD16 downregulation in aged neutrophils.
Dual labeling was performed on neutrophils cultured for 24 h by
incubating the cells with 3G8 MoAb, followed by anti mouse IgG1/PE, and
then by FITC-conjugated W6/32. Histograms are plotted with PE
fluorescence on the x axis and with FITC fluorescence on the
y axis. Relative percentages of the gated populations are
indicated. A representative experiment of the two performed is shown.
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Figure 4. Time course of Class I expression and Annexin V binding in aging
neutrophils. Dual labeling was performed on freshly isolated
neutrophils (A), and after 18 (B) and 24 (C) h of culture. After
indirect immunofluorescence with W6/32 and anti-mouse IgG2a/PE was
performed, cells were incubated with FITC-conjugated Annexin V.
Histograms in the left column are plotted with forward scatter
on the x axis and with side scatter on the
y axis. Histograms in the right column are plotted with FITC
fluorescence on the x axis and with PE fluorescence on the
y axis. The gating set for analyzing the cells in the right
column is indicated in the left column. For each histogram, the
relative percentage of the cells in the respective field is indicated
in the right column. A representative experiment of the three performed
is shown.
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Figure 5. Evaluation of apoptosis in neutrophils expressing normal levels of
Class I and low levels of Class I. After 24 h of culture, 2 x 107 neutrophils were labeled with FITC-conjugated W6/32
MoAb (upper panel). By means of a fluorescence-activated cell sorter,
the cells were divided into two subpopulations of low expression of
Class I (A) and normal expression of Class I (B). Each sorted sample
was then divided into three aliquots for measuring apoptosis. Propidium
iodide incorporation was evaluated after cold-ethanol fixation and
overnight incubation with propidium. DNA fragmentation was checked by
gel electrophoresis of DNA extracted from the two samples. Lanes 1
indicate the relative sample; lanes 2 indicate the molecular weight
markers. Cell morphology was studied on cytocentrifuge preparations
stained with May Grunwald-Giemsa. A representative experiment of the
two performed is shown. Neutrophils (54%) showed hypodiploid DNA
content, and 64% displayed morphological features of apoptosis in the
sorted sample, expressing low levels of Class I (A), compared with 12%
and 31%, respectively, in the sorted sample expressing normal levels
of Class I.
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Figure 6. Effect of drugs preventing apoptosis on Class I downregulation. Freshly
isolated neutrophils were incubated for 24 h in the absence (A) or
presence of 10 ng/mL GM-CSF (B) or 1 mM cAMP (C). Afterward, Class I
expression was evaluated by indirect immunofluorescence with the W6/32
mAb, and the amount of apoptosis was measured by the Annexin/FITC
technique. The percentage of apoptotic cells was 50.4% in A, 26.5% in
B, and 25.8% in C. A representative experiment of three that were
performed with GM-CSF and two that were performed with cAMP is shown.
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Figure 7. Effect of surface Class I ligation on age-related apoptosis in
neutrophils. Freshly isolated neutrophils were incubated for 24 h
in medium alone (hatched bars), medium plus F(ab')2
fragments of the 3G8 mAb (open bars), and medium plus
F(ab')2 fragments of the W6/32 mAb (solid bars). The
percentages of living and apoptotic cells were measured in each sample
by evaluating cell morphology, Annexin V/FITC binding, and propidium
iodide incorporation. Values are expressed as % living cells/%
apoptotic cells. Results shown are the mean determination ± 1
SD from two separate experiments for morphology and
propidium iodide and from four separate experiments for Annexin V. In
the morphology and propidium iodide assays: medium alone vs. medium
plus W6/32, p < 0.05; medium alone vs. medium plus
3G8, p > 0.05. In Annexin V assay: medium alone vs.
medium plus W6/32, p < 0.01; medium alone vs. medium
plus 3G8, p > 0.05. One-way analysis of variance
(ANOVA) with Bonferroni multiple comparison post test was used.
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This in vivo apoptosis is mimicked in vitro by the spontaneous apoptosis displayed by neutrophils in culture [16 , 17 , 19 ]. In this study, using the experimental conditions that induce apoptosis, we observed a marked reduction in Class I expression. In our experiments, all basic conditions that could account for this finding, such as contamination by cells other than granulocytes, the presence of interfering necrotic cells or cellular debris, and binding to membrane Fc receptors, have been ruled out. Similarly, the hypothesis that surface Class I modulation could be strictly related to cell activation could not be responsible, because a powerful activator like PMA did not alter the membrane expression of Class I molecules. Because the subpopulation with reduced expression increases over the time, a mechanism involving the cells progressively could be responsible.
As a matter of fact, our data suggest a close relationship between Class I downregulation and neutrophil senescence. The temporal changes in Class I expression closely resemble those shown by CD16 [16 ], a membrane molecule whose expression is reduced in aging neutrophils [16 , 17 ]. Notably, in our experiments, CD16 is more downregulated than HLA Class I. The difference in the degree of downregulation, together with a marked individual variability in the time course of age-related apoptosis, could account for the fact that Dransfield and coworkers [16 ] failed to find Class I downregulation in their system.
CD16 and Class I are not the only surface molecules whose expression is modified in aging neutrophils. Actually, several changes occur at the membrane level in these cells, with some molecules being upregulated, some being downregulated, and some remaining unchanged [16, 17, 28, and present paper]. These findings suggest that reshuffling the membrane molecules in polymorphonuclear neutrophils (PMNs) on the way to death is not only a precisely regulated, but also an apparently specific, phenomenon. At present, a fine analysis of the reasons as to why this surface reorganization occurs is lacking. Thus, it may only be hypothesized that it occurs because of particular behaviors of the different molecules, probably related to distinctive functions. From our experimental data, initial conclusions can be drawn about the functions of CD16 and Class I only.
Neutrophils could downregulate the expression of these two molecules to
ignore the signals delivered through them perturbing their orderly
progression to apoptosis. Apoptosis is crucial for the removal of aged
granulocytes. Neutrophils that have started their cell death program
exist in a state of functional isolation from the external milieu,
displaying a global impairment in their functions, such as chemotaxis,
secretion, and metabolic burst [19
, 29
].
This condition is retained for more than 24 h before the
disintegration and release of their contents, a period long enough to
allow macrophages to be recruited and then to phagocytose the apoptotic
neutrophils [30
, 31
]. Thus, to enter and
maintain this standby condition, neutrophils have to switch off all
signals that can induce activation or prevent apoptosis. Downregulation
of CD16 in aging neutrophils is probably directed at this purpose. Its
ligation results in granule release, reorganization of actin
microfilaments, and priming of FcRII-mediated phagocytosis, events
that are associated with neutrophil activation and effector cell
function [32
, 33
]. Thus, it is reasonable
that a marked reduction in CD16 expression may render the cells less
sensitive to stimuli that trigger effector functions. In fact, removal
by phospholipase C of greater than 80% of surface CD16, a threshold
well above the reduction observed in aged neutrophils, abolishes
chemotactic responses to formyl-Met-Leu-Phe (fMLP) [34
].
A similar model could be used to explain the reduction in Class I expression on aged neutrophils. Indeed, Class I downregulation could render neutrophils less sensitive to signals preventing apoptosis delivered by this molecule. From this perspective, we have analyzed the relationship linking Class I to age-related apoptosis. As shown, Class I downregulation parallels apoptosis. Even more interestingly, conditions inhibiting apoptosis (such as GM-CSF and cAMP) inhibit downregulation also. To further confirm the close relationship between the two phenomena, the effect of HLA class I ligation on programmed cell death was also studied. For this purpose, we used the Fab fragment of the W6/32 mAb, which recognizes a monomorphic determinant on all human HLA-A, -B, and -C subclasses and has been used widely, either soluble or cross-linked, in experiments on Class I ligation [2 3 4 5 , 7 8 9 10 11 ]. As shown, ligation of Class I molecules delayed neutrophil progression to apoptosis markedly. Similar evidence was not achieved by ligation of CD16 molecules.
To our knowledge, the "antiapoptotic" effect of HLA Class I ligation represents the first evidence so far of signal transduction via Class I molecules in neutrophils. This effect was unexpected partially, and only speculative explanations can be proposed. At present, two natural ligands of HLA Class I molecule have been described. One is the TcR/CD8 complex, of which two ways for neutrophilic Class I ligation to occur should be considered. One could occur through the specific recognition of a nonself peptide, and the other is based on low-affinity binding to Class I molecules in the absence of the antigen. The latter does occur peripherally and probably plays, in lymphocytes, a role in the maintenance of T cell memory [35 36 37 38 ]. At present, our data do not allow us to arrive at any conclusions on the significance or relevance of the two mechanisms. The other natural ligand for Class I molecule is represented by Killer Receptors, either activating or inhibitory, expressed by natural killer (NK) cells [39 ]. No data are available, to our knowledge, on the functional relationship between NK cells and aged neutrophils. However, from a speculative point of view, an increased susceptibility of neutrophils having downregulated Class I to NK cell lysis should result in the spreading of harmful substances by neutrophils. Also of importance to consider is that Class I has been shown to be structurally and/or functionally associated with peptide hormone receptors, some of which are also expressed by neutrophils, such as receptors for IL-2, insulin, insulin-like growth factor, and glucagon [40 41 42 43 44 45 46 47 ]. Thus, Class I molecules could be involved in signal transduction by modifying or fine-tuning signaling via other cell-surface molecules [11 , 48 ].
Received January 4, 2000; revised June 19, 2000; accepted June 20, 2000.
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3 domain of Class I MHC Science 252,1424-1427RIII) expression J.Immunol. 153,1254-1263[Abstract]RIII and acquire annexin V binding sites during apoptosis in vitro Blood 85,532-540 receptor III induces actin polimerization in human neutrophils and primes phagocytosis mediated by Fc receptor II J. Immunol. 146,997-1004[Abstract]RIIIB and formyl peptide receptors are functionally linked during formyl-methionyl-leucyl-phenylalanine-induced chemotaxis J. Immunol. 149,989-997[Abstract]This article has been cited by other articles:
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