FEMS Immunology and Medical Microbiology 34 (2002) 135^138
www.fems-microbiology.org
E¡ect of phage therapy on the turnover and function of
peripheral neutrophils
Beata Weber-Dabrowska, MichaI Zimecki , Marian Mulczyk, Andrzej Go¤rski
Institute of Immunology and Experimental Therapy of the Polish Academy of Science, Rudolfa Weigla 12, 53-114 WrocIaw, Poland
Received 18 February 2002; accepted 23 July 2002
First published online 12 September 2002
Abstract
The aim of this investigation was to establish the impact of phage therapy on the turnover and function of circulating neutrophils in 37
patients with suppurative bacterial infections. We determined the levels of circulating neutrophils and their precursors before therapy,
after 3 weeks of therapy, and at a distant time interval (3 months) following the beginning of therapy. In addition, we measured the ability
of neutrophils to phagocytize Staphylococcus aureus in vitro. Eight healthy blood donors served as a control group. The results showed
that, among the studied parameters, the significant changes involved neutrophil precursor count and the ability of neutrophils to
phagocytize bacteria. The percentage of neutrophils in patients before therapy was lower than in healthy donors (mean 58.0, versus 61.4).
This value dropped further in patients after 3 months of following the therapy (mean 55.6). The content of neutrophil precursors, on the
other hand, was lower in healthy donors than in patients before therapy (mean 2.5, versus 3.8). After 3 weeks of the therapy and after
3 months, the levels of neutrophil precursors were significantly higher (mean 4.8 and 4.9, respectively) than in control donors. The
phagocytic index was lower in patients before therapy than in control donors (mean 66.3, versus 70.1) and decreased further after 3 weeks
of therapy (mean 59.0) and after 3 months (mean 59.6). The results of this investigation indicate that successful phage therapy accelerates
the turnover of neutrophils, accompanied by a decrease in their ability to phagocytize bacteria.
; 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Phage therapy; Neutrophil precursors ; Phagocytosis ; Staphylococcus aureus
1. Introduction
The increasing resistance of pathogenic bacteria to antibiotics has led to renaissance in phage therapy [1]. Phage
therapy has proved to be e¡ective in combating suppurative bacterial infections resistant to antibiotics and in providing long term immunity [2]. Our recent investigation
also revealed that the successful phage therapy was associated with a normalization of cytokine production in cultures of peripheral blood cells of patients [3].
In the present investigation we turned our attention to
neutrophils, the major and most e⁄cient phagocytes in
circulating blood. Neutrophils are among the cells with a
very short lifespan and a very high daily output from bone
marrow [4^6]. Beside their ability to phagocytize and kill
bacteria, neutrophils secrete a number of in£ammation
mediators [7^10] and exhibit regulatory functions [11].
The aim of this study was to determine the in£uence of
the phage therapy on the levels of neutrophils and their
precursors in the circulation of patients with suppurative
infections. In addition, the ability of neutrophils to ingest
Staphylococcus aureus was evaluated.
2. Materials and methods
2.1. Patients and healthy donors
* Corresponding author. Department of Experimental Therapy, Institute of Immunology and Experimental Therapy, R. Weigla 12, 53-114
WrocIaw, Poland, Tel.: +48 (71) 3732274; Fax: +48 (71) 373 2587.
E-mail address : [email protected] (M. Zimecki).
42 patients (19 women and 23 men) were enrolled into
study. Patients su¡ered from: rhinitis (4), furmucolosis (4),
sinusitis (4), post-trauma infections (3), infections of the
reproductive tract (2), chronic bronchitis (1), urinary tract
infections (1), perianal abscess (1), dermal infections (1),
and bilateral otitis (1).
0928-8244 / 02 / $22.00 ; 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 9 2 8 - 8 2 4 4 ( 0 2 ) 0 0 3 7 5 - 9
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B. Weber-Dabrowska et al. / FEMS Immunology and Medical Microbiology 34 (2002) 135^138
The bacterial strains isolated from the patients were the
following: Staphylococcus aureus (32), Staphylococcus epidermidis (6), Escherichia coli (5), Klebsiella oxytoca (1),
Morganella morganii (1), Staphylococcus simularans (1),
Staphylococcus hominis (1), Staphylococcus saprophyticus
(1). Homogenous infection was detected in 35 cases and
mixed infection in seven cases.
The group of healthy donors constituted of eight subjects (six women, 32^53 years old and two men, 36^74
years old).
Treatment of patients with phage preparation was performed as described elsewhere [12], i.e. by oral application
of 5 ml of phage lysate 3U daily for 3 weeks.
2.2. Phagocytosis
Samples of venous blood (2 ml), to which sodium citrate
was added, were centrifuged for 10 min at 2000 r.p.m.,
and a bu¡y coat was collected. The cells were washed
3U with Hanks’ medium, and the leukocytes counted.
To 100 Wl of heat-inactivated autologous serum, 100 Wl
of Staphylococcus aureus, strain 209 P suspension, and
100 Wl of leukocyte suspension were added. The number
of bacteria was assessed by means of optical density using
the appropriate standard. The ratio of bacteria to leukocytes was 100:1. The mixture was incubated for 45 min at
37‡C with occasional shaking. The cells were then vigorously mixed using a syringe to obtain a single-cell suspension. 5 Wl of the suspension was applied onto a microscopic glass and a smear was prepared. The smears were dried
and, on the next day, ¢xed with 96% methyl alcohol for
10 min, then dried and stained with Manson blue. The
preparations were examined under a microscope (800U
magni¢cation, in immersion oil). 100 neutrophils were
counted and the numbers of ingested bacteria in each
cell were determined. The results are shown as the phagocytic index, i.e. the mean number of bacteria per phagocyte. The mean value from two such samples (smears) for
each blood donor was calculated.
Fig. 1. The content of neutrophils (A) and neutrophil precursors (B) in
the peripheral blood of healthy donors and patients subjected to phage
therapy: before therapy (1), after 3 weeks of therapy (2), and 3 months
after the beginning of therapy (3).
2.3. Morphology of blood cells
A drop of heparinized blood was applied to a microscope slide and a smear was made. After drying, the preparation was treated with May^Gru«nwald stain and then
with Giemsa reagent. The cells were counted (di¡erentiated) at a magni¢cation of 800U in immersion oil. Up to
200 cells were counted. The results are presented as a
Table 1
Comparison of white blood cell type composition in patients subjected to phage therapy with healthy donors
Control
donors
Patients
before
therapy
After 3
weeks
After 3
months
Neutrophil
precursors
Neutrophils
Eosinophils
Basophils
Monocytes
Lymphocytes
mean T
P
S.E.M.
mean T
P
S.E.M.
mean T
P
S.E.M.
mean T
P
S.E.M.
mean
T
P
S.E.M.
mean T
P
S.E.M.
2.54
0.38
61.36 1.67
2.72
0.76
0.18
0.12
0.45
0.20
32.72 1.53
3.82
0.38
NS
57.97 1.27
NS
3.10
0.36
NS
0.70
0.11
6 0.05
0.45
0.94
NS
34.15 1.16
NS
4.80
0.42
6 0.001 57.02 1.19
NS
3.57
0.43
NS
0.52
0.10
NS
0.45
0.16
NS
33.47 1.10
NS
4.82
0.55
6 0.05
6 0.001 3.19
0.39
NS
0.40
0.09
NS
0.17
0.06
NS
34.16 1.06
NS
55.61 1.08
The data are presented as percentage of mean values T S.E.M.
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percentage of : lymphocytes, eosinophils, basophils, monocytes, neutrophils and their immature forms.
2.4. Statistics
For the determination of changes in the studied parameters, the Mann^Whitney U-test was applied. The data are
presented as median values and quartiles (the ¢gures) or
mean values (the table) accompanied by standard errors.
The changes were regarded as signi¢cant when P 6 0.05.
3. Results and discussion
The determination of the basic blood cell types in the
peripheral blood of patients, including neutrophils, lymphocytes, eosinophils, basophils and monocytes, revealed
that signi¢cant changes, compared with the di¡erential
leukocyte counts of healthy donors, involved only neutrophils and neutrophil precursors (metamyelocytes and band
cells) (Table 1). In addition, the di¡erences in the total
white cell count between the patients (at the respective
time intervals) and control donors were not statistically signi¢cant (6369 T 350, 6461 T 296, 6097 T 323 and
5964 T 350). In Fig. 1A, B, the levels of neutrophils and
neutrophil precursors before and after the phage therapy
compared with the levels recorded in control donors are
shown. The percentage of neutrophils was slightly lower in
the patients before therapy than in the healthy donors.
That level did not change after 3 weeks of therapy, but
after 3 months the level of neutrophils dropped signi¢cantly (P 6 0.05) compared with the control value. On
the other hand, the level of neutrophil precursors, similar
in the patients before therapy to that in the healthy individuals, was signi¢cantly higher after 3 weeks and
3 months (P 6 0.05) following initiation of phage therapy.
Interestingly (Fig. 2), the ability of neutrophils to
phagocytize bacteria was lower in patients before therapy
than in healthy blood donors, this function further decreasing following therapy (P 6 0.05). This decreased abil-
137
ity of neutrophils from the patients to phagocytize Staphylococcus aureus compared with healthy donors could be
explained by the e¡ects of prolonged, ine⁄cient antibiotic
therapy preceding phage therapy. The phenomenon of a
decreased [13,14], unchanged [15,16] or increased [17] ability of neutrophils to phagocytize or kill bacteria has been
described in patients treated with antibiotics.
We did not observe any correlation between the outcome of the therapy and the observed changes in the degree of phagocytosis (complete healing n = 19, distinct
improvement n = 8, and partial improvement n = 10).
However, a kind of regulation in the magnitude of this
parameter was noted when the patients were classi¢ed as
those with initially low and high abilities to phagocytize
bacteria (not shown). The decreasing ability to phagocytize bacteria in the recovering patients may be associated
with a diminished necessity to eliminate bacteria. In addition, the higher percentage of immature neutrophils, unable to ingest bacteria, in the circulation may contribute to
this phenomenon. Careful analysis of individual cases revealed that after 3 months the neutrophils began to regain
their phagocytic capacity. In other studies (with surgical
patients), a decreased ability to phagocytize Staphylococcus aureus was compensated by an elevated number of
neutrophils 4 days after surgery [18].
The most important observation of this investigation
was that the phage therapy accelerated the turnover of
neutrophils. This was evidenced by a signi¢cant increase
in the level of the immature forms of neutrophils accompanying the decrease of mature neutrophils.
The increased percentage of immature neutrophils may
result from the following scenario. First, the action of
phages causes bacterial cell destruction and induction of
proin£ammatory cytokines by bacterial products, including
interleukin 8, leading to degranulation of neutrophils and
lactoferrin release. Lactoferrin has a property of inducing
several cytokines, including colony-stimulating factors,
which recruit neutrophil precursors from the bone marrow.
Such a mechanism of increased turnover of neutrophils,
involving lactoferrin as a ‘demand’ signal for colony-stimulating factor production, was proposed by Rich [19].
In summary, the presented data show that the phage
therapy elicits in patients changes in neutrophil turnover
and function. Although the decreased level of mature neutrophils during therapy may not seem bene¢cial, it was
clearly associated with recovery. It is possible, however,
that the prolonged stimulation of the neutrophil turnover
observed in this study may be advantageous for patients
by enhancing the ¢rst line of defence.
Acknowledgements
Fig. 2. The phagocytic index of neutrophils derived from peripheral
blood of healthy donors and patients subjected to phage therapy: before
therapy (1), after 3 weeks of therapy (2), and 3 months after beginning
of therapy (3).
This work was supported by Grant 4PO5B01218 from
the State Committee for Scienti¢c Research (KBN), Poland.
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