* MRC Centre for Immune Regulation, Birmingham University Medical School, Birmingham, United Kingdom; and Departments of
Geriatric Medicine and
Medicine, University Hospital, Birmingham, United Kingdom
Correspondence: Dr. J. M. Lord, MRC Centre for Immune Regulation, Birmingham University Medical School, Birmingham B15 2TT, UK. E-mail: J.M.Lord{at}bham.ac.uk
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Key Words: immunesenescence • aging • neutrophil-function
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Neutrophils mediate the immediate host response to bacterial and fungal infections, which are largely responsible for the higher rates of mortality and morbidity in the elderly population [8 ]. Neutrophils are short-lived (half-life, 12–18 h), post-mitotic granulocytic cells that are produced in vast numbers (1–2x1011 per day) in the bone marrow. Several studies have shown that neutrophil numbers in the blood [9 , 10 ] and neutrophil precursors in the marrow [9 ] are not lowered in the healthy elderly; thus, neutrophil supply does not appear to be a major source of immune compromise in the innate response to bacterial infection. Similarly, the chemotactic responses of neutrophils do not appear to be significantly affected by age. In vitro studies of chemotaxis have shown migratory responses of neutrophils from healthy, elderly subjects to be unaltered [2 , 11 ], and adhesion of neutrophils from elderly subjects to endothelium was also unchanged [2 , 12 ]. The two remaining potential sources of compromised neutrophil bactericidal function are phagocytosis and bactericidal mechanisms such as superoxide generation and degranulation. These aspects of neutrophil function have been the focus of this study.
The few studies of neutrophil phagocytosis in the elderly are consistent in that they have shown an adverse effect of age on neutrophil phagocytic ability [13 14 15 ]. The reduced response of neutrophils to Staphylococcus aureus [15 ] is clinically important because of the increased susceptibility to this pathogen in elderly subjects [8 ]. However, only one study has looked at the number of neutrophils with phagocytic capacity and the number of bacteria ingested per neutrophil [15 ], the phagocytic index. This is important, because a simple reduction in cells able to phagocytose microbes could be counteracted by increased production and recruitment of neutrophils to sites of infection. Adequate recruitment of neutrophils to sites of infection would be significantly offset and the ability of the elderly to resolve an infection compromised if the neutrophils recruited had a reduced phagocytic capacity.
In this study, we have measured phagocytic index in neutrophils from
healthy, young and elderly donors as well as measuring superoxide
generation in response to formyl-Met-Leu-Phe (fMLP). Our data show
reduced phagocytic index in neutrophils from elderly donors, with no
reduction in superoxide generation. Phagocytosis is initiated by the
interaction of specific receptors on the surface of the neutrophil with
particulate ligands on the microbe. The key receptors inducing
phagocytosis of bacteria by neutrophils are those for the Fc region of
immunoglobulin G (IgG; Fc
RIII/CD16 and FcRII/CD32) and for the
complement molecules C3b (CD35/CR1) and C3bi (CD11b/CR3), which are
bound to the surface of the microbe. Analysis of expression of
receptors for complement (CD11b) and Ig (CD16) on neutrophils revealed
that CD16 expression was significantly reduced in the elderly. This
loss of CD16 could be a major factor contributing to reduced neutrophil
function and increased susceptibility to bacterial infections in the
elderly. Indeed, when we examined CD16 expression in elderly patients
with bacterial infection and neutrophilia, we found that CD16
expression remained low. This would suggest that neutrophils released
recently from the bone marrow during infection also have reduced CD16
expression and that the altered expression of CD16 on neutrophils may
arise in the bone marrow of the elderly.
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Neutrophil isolation
Human neutrophils were isolated from venous blood, taken by
venepuncture from consenting volunteers, on Percoll (Sigma Chemical
Co., St. Louis, MO) density gradients as previously described
[17
]. Briefly, blood was drawn and dispensed immediately
into 50 ml sterile polypropylene centrifuge tubes containing 30 mg
ethylenediaminetetraacetate (EDTA) and mixed gently. The blood was then
mixed with Hespan (Fresenius Medical Care, Lexington, MA) at a ratio of
1:7 ml blood to sediment erythrocytes. The leukocyte fraction was
removed, mixed 1:1 with sterile phosphate-buffered saline (PBS), then
layered onto Percoll gradients consisting of 5 ml 54% Percoll and 2.5
ml 78% Percoll. Gradients were spun for 25 min at 1100 rpm (MSE
Centaur II, Fisher Scientific, Loughborough, UK) at room temperature.
The lower granulocyte band was then removed, washed in sterile PBS, and
resuspended in RPMI 1640 medium (Invitrogen Life Technologies,
Carlsbad, CA) containing 10% fetal calf serum (Sera Laboratories
International, Crawley, U. K.), 2 mM glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin. Neutrophil preparations were
assessed for purity by differential staining using a commercial Giemsa
staining kit (Diff-kwik, Baxter Healthcare, Deerfield, IL) and were
routinely >95% neutrophils.
Measurement of superoxide generation in response to fMLP
Neutrophil respiratory burst in response to fMLP was determined
in isolated neutrophils by measuring the generation of superoxide in a
lucigenin-based assay [18
]. The cells were washed once
in RPMI 1640 medium, once in PBS, and resuspended in Hanks’ balanced
saline solution (HBSS) 1% bovine serum albumin (BSA) buffer at 2 x 106 cells/ml. The cell suspension (100 µl) was added
to plastic chemiluminometer tubes followed by 20 µl lucigenin (2.5 mM
in HBSS; Sigma Chemical Co.) and incubated at 37°C in a water bath
for 5 min. Two control tubes were used in each assay, one included no
stimulus (fMLP) and the other, no cells. The assay was initiated by the
addition of 100 nM fMLP (Sigma Chemical Co.) and superoxide generation
measured over 10 min, as the release of light from lucigenin excited by
reactive oxygen species. Results were expressed as integral counts per
minute, determined from the area under the curve of the reaction for
the first 10 min.
Assessment of phagocytosis of fluorescein isothiocyanate
(FITC)-labeled Escherichia coli
Phagocytosis of E. coli by neutrophils was measured
using a commercial fluorescence-based kit (Phagotest, Orpegen,
Germany), according to the manufacturer’s instructions. Briefly, 100
µl venous blood was incubated at 4°C for 10 min, then mixed with 20
µl of a suspension of FITC-labeled E. coli
(1x109 bacteria/ml), and opsonized with whole human serum.
Control tubes were kept at 4°C to monitor adherence of the labeled
bacteria to neutrophils in the absence of uptake. The phagocytosis test
tubes were incubated at 37°C for 10 min. Samples were then placed
immediately at 4°C and mixed with FITC quench-buffer to eliminate
fluorescence from bacteria sticking to the neutrophil cell surface. The
blood was then washed twice in PBS, the erythrocytes were lysed, and
the leukocytes were fixed for 20 min with the lysing/fixation solution
provided at room temperature. The cells were then washed twice and
finally incubated with a nuclear counter-stain for 10 min at 4°C. The
assay was performed in duplicate and analyzed by flow cytometry, gating
against the level of green fluorescence (FL1) seen in the 4°C control
sample. Neutrophils were distinguished from other leukocytes by gating
on forward- and side-scatter, and any remaining E. coli were
excluded using the red fluorescent (FL2) stain to identify cells having
diploid DNA. Ten-thousand events were measured to assess the percentage
of neutrophils that had phagocytosed E. coli and the mean
fluorescence intensity (MFI)—a measure of the number of bacteria taken
up per cell. Data were then expressed as the neutrophil phagocytic
index: Phagocytic index = % phagocytic neutrophils x MFI.
Analysis of cell-surface molecules by flow cytometry
Neutrophils were isolated as described above and resuspended at
2 x 106 cells/ml. Cells (2x105) were
then washed and incubated with blocking buffer (PBS containing 10%
human serum), followed by incubation with FITC-conjugated monoclonal
antibodies (mAbs) to CD11a, CD11b, and CD16 (Dako, Bucks, UK) at
saturating concentrations at 4°C. Cells were washed and fixed in PBS
containing 4% paraformaldehyde for 10 min at 4°C. Fluorescence was
analyzed by flow cytometry (Coulter Epics XL, Luton, UK), and 5000
events were collected per sample.
Phagocytosis versus CD16 expression
Whole blood (100 µl) was kept at 4°C for 10 min prior to
assessment of phagocytosis of E. coli and CD16 expression in
a dual-labeling protocol. A cooled FITC-labeled E. coli
suspension (Orpegen Pharma, Heidelberg, Germany) was added to the blood
and incubated for 10 min at 37°C, and a control was kept at 4°C.
The tubes were then placed immediately at 4°C to stop further
phagocytosis. For CD16 analysis, the blood was then incubated with
saturating levels of unconjugated anti-CD16 mAb (Dako) for 15 min at
4°C. The blood was then washed and incubated at 4°C for 15 min with
a phycoerythrin (PE)-conjugated rabbit anti-mouse IgG antibody.
Controls were treated with mouse IgG followed by anti-mouse IgG
antibody. All samples were then treated with FITC quench, washed twice
in PBS, and resuspended in lysis/fixation buffer (Orpegen Pharma). The
samples were analyzed by flow cytometry to give a MFI of CD16 labeling
and phagocytic index.
Opsonization efficiency of serum and effect of IgG depletion on
E. coli phagocytosis assay
To determine whether serum from elderly donors was able to
opsonize E. coli as efficiently as serum from young donors,
unopsonized FITC-labeled E. coli were combined with serum
from young and old donors, and the ability of neutrophils from a single
donor to phagocytose the E. coli was determined. Unopsonized
(40 µl) FITC E. coli (Orpegen Pharma) was washed twice in
PBS, incubated at 37°C for 40 min, and resuspended in 800 µl
complement fixation buffer (ICN Pharmaceuticals, Costa Mesa, CA) and
100 µl donor serum. The E. coli were then washed twice in
PBS and resuspended at 2 x 107 per 20 µl in PBS.
Neutrophils (100 µl) at 2 x 106 cells/ml were then
incubated for 2 h in RPMI 1640 containing 2% BSA and were then
combined with 50 µl each donor serum and 20 µl E. coli
opsonized with the same serum. The phagocytosis assay was then
performed as described above.
To determine the contribution of immunoglobulin binding to phagocytosis of E. coli in the assay used here, serum depletion was performed using protein A/G sepharose (Santa Cruz Biotechnology, Santa Cruz, CA). The depleted serum was passed through a 0.2 µm filter (Gelman Sciences, Ann Arbor, MI) and used in the phagocytosis assay as described above. Depletion of IgG was confirmed by Western blotting using a horseradish peroxidase (HRP)-conjugated anti-IgG antibody (Sigma Chemical Co.) and enhanced chemiluminescence (ECL) detection system (Amersham, Arlington Heights, IL).
Statistics
Differences among groups were determined using a
t-test, and a P value of <0.05 was taken as a
significant difference between means. Correlations were determined
using Pearson’s correlation analysis.
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Figure 1. Neutrophil superoxide response to fMLP. Neutrophils were isolated from
23 healthy, young (23–35 years) and elderly (>65 years) volunteers
and incubated for 10 min with fMLP as described in Materials and
Methods. Superoxide generation for each volunteer is shown, and the
median for each group is marked.
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Figure 2. Phagocytosis of E. coli by neutrophils of young and elderly
donors. Phagocytosis of E. coli by neutrophils was measured
in whole blood using a commercial kit (Phagotest, Orpegen Pharma).
Blood was incubated with FITC-labeled E. coli for 30 min,
and uptake of bacteria was determined by FACS analysis according to the
manufacturer’s instructions. Data for the phagocytic index (% of
neutrophils that had phagocytosed E. coli x MFI) for
neutrophils from 11 healthy, young (23–35 years) and elderly (>65
years) donors are shown. The median for each population is marked.
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Figure 3. Opsonization of E. coli with serum from young and elderly
donors. Serum from five healthy, young and elderly donors was incubated
with unopsonized FITC-labeled E. coli prior to addition of
neutrophils from a single, young donor. Phagocytic index was measured
as described in Materials and Methods, and data shown are mean ±
SD.
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Figure 4. Effect of age on expression of CD11b and CD16. Neutrophils from
healthy, young (23–35 years) and healthy, elderly (>65 years)
volunteers were immunostained with FITC-conjugated mAbs to (A) CD11b
(n=17) or (B) CD16 (n=23). Neutrophils from
elderly (>65 years) patients with bacterial infections were also
assessed for CD16 expression (n=19). Fluorescence was
analyzed by flow cytometry, and data are expressed as MFI for each
sample. The median value is shown for each age group.
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Figure 5. Effect of removal of IgG on neutrophil phagocytosis. Serum from young
and old, healthy donors was treated with protein A/G agarose to remove
IgG. (A) The depleted serum was used to opsonize FITC-labeled E.
coli prior to incubation with isolated neutrophils and measurement
of phagocytic index. Data shown are the mean of triplicate values from
a single experiment representative of two performed. (B) Depletion of
IgG was checked by Western blotting of serum samples (5–20 µl) using
an antihuman IgG antibody.
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Figure 6. Effect of CD16 expression on neutrophil phagocytosis. Neutrophils from
six healthy volunteers were incubated with FITC-labeled E.
coli prior to fixation and staining for CD16 with a PE-conjugated
anti-CD16 antibody. (A) Neutrophils were analyzed for CD16 expression
and uptake of E. coli by FACS analysis, and two
representative FACS dot plots and single-channel histograms are shown.
(B) Data for CD16 expression (MFI) and phagocytic index were calculated
and are shown for each sample. The linear regression value for the plot
was r = 0.83, with P < 0.05.
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The data concerning superoxide responses are in agreement with previous publications showing a normal response to fMLP in the elderly [12 , 20 ]. However, in contrast to the data regarding fMLP, studies concerning superoxide generation in response to particulate stimuli have indicated a reduced response to these agents in the elderly. Wenisch and coworkers [15 ] have shown that superoxide generation was decreased in response to S. aureus but not to E. coli, an observation with particular clinical relevance because of the reduced ability of the elderly to resolve infection by gram-positive bacteria [8 ]. We also found that superoxide generation in response to E. coli was not affected by age (unpublished results). It is possible that superoxide responses to E. coli, which involve binding of lipopolysaccharides by CD14 [21 ], may be unaffected by age, although responses to gram-positive bacteria, which are more dependent on complement and Fc-receptor ligation, are attenuated. Indeed, the Fc receptor-mediated superoxide response has been shown already to be reduced significantly in the elderly [22 ], again identifying attenuation of Fc-mediated responses as a significant factor in age-related neutrophil functional decline.
The notion that altered Fc responses contribute significantly to the decline in neutrophil function with age is supported by our data concerning phagocytosis of E. coli. The Fc-gamma receptor clearly plays a key role in dictating the level of the phagocytic response in neutrophils, because removal of IgG markedly reduced phagocytic index. In addition, the level of CD11b was essentially unaltered in the elderly, indicating that loss of CD16 was a specific age-related effect rather than a reflection of a general decline in surface-protein expression. The decline in CD16 levels with age, shown here, provides the first data on the molecular events underlying attenuated Fc-mediated phagocytosis in the elderly.
Expression of CD32, the other low-affinity Fc-gamma receptor present on resting neutrophils, has not been studied here. CD16 is expressed at 4–5 times the level of CD32 and is thought to play the dominant role in binding IgG on neutrophils [23 ]. CD64, the high-affinity Fc-gamma receptor, is only expressed on primed neutrophils [24 ]. Therefore, loss of CD16 would be expected to reduce phagocytic efficiency, and this is borne out by our data.
Although reduced phagocytosis in neutrophils from elderly subjects has been shown previously [13 14 15 ], the molecular basis of this decline has not been established. Our data indicate that reduced phagocytosis in the elderly is intrinsic to the neutrophil itself, because serum from elderly donors was able to opsonize bacteria efficiently. Previous studies demonstrating that immunoglobulin and complement levels were within the normal range in the elderly [2 ] are in agreement with this conclusion. Emmanuelli et al. [14 ] showed that phagocytosis of unopsonized bacteria was not reduced in the elderly, suggesting that receptors for innate recognition of bacterial components (e.g., CD14) were not affected by age and that the reduced response of neutrophils to opsonized E. coli was likely to be mediated by a reduced expression of the relevant receptors or as a result of compromised signalling function. The data shown here support a role for reduced Fc-gamma receptor expression in neutrophil functional decline in the elderly. Currently, we are investigating whether there is an effect of aging on Fc-receptor signals involved in phagocytosis.
To determine whether reduced expression of CD16 had arisen in the circulation of the elderly or was already present on neutrophils as they were released from the bone marrow, we measured CD16 expression in the elderly during neutrophilia. Bacterial infections increase the production and release of neutrophils from the bone marrow into the circulation, an important factor in combating infection. It is interesting that despite a profound neutrophilia in the elderly patients, CD16 expression remained low, suggesting that the alteration in CD16 expression arises in bone marrow. Moreover, although the elderly can mount an adequate neutrophilia, the neutrophils produced to combat infection also have reduced CD16, limiting their ability to combat the infection. In conclusion, we have shown that phagocytic capacity is reduced as humans age, and reduced expression of the Fc-gamma receptor CD16 may contribute to this decline. Preliminary data suggest that CD16 expression is already low on neutrophils released from the bone marrow. Because phagocytosis of bacteria by neutrophils is crucial in the early phase of infection, if this function is compromised in the elderly, it will lead to a reduced ability to combat bacterial infections.
Received March 12, 2001; revised July 23, 2001; accepted August 2, 2001.
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