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International Immunopharmacology 3 (2003) 853–864
Bartonella quintana lipopolysaccharide effects on leukocytes,
CXC chemokines and apoptosis: a study on the human whole blood
and a rat model
Giovanni Materaa,*, Maria Carla Libertoa, Angela Quirinoa, Giorgio Settimo Barrecaa,Angelo Giuseppe Lambertia, Michelangelo Iannoneb, Eliana Mancusoc,
Ernesto Palmac, Francesco Antonio Cufaric, Domenicantonio Rotirotib,c, Alfredo Focaa
a Institute of Microbiology, Department of Medical Sciences, Faculty of Medicine, University of Catanzaro,
Via T. Campanella 115, I-88100 Catanzaro, Italyb ISN-CNR Section of Pharmacology, Roccelletta di Borgia, Catanzaro, Italy
cFaculty of Pharmacy, University of Catanzaro, Catanzaro, Italy
Received 2 August 2002; received in revised form 3 December 2002; accepted 20 February 2003
Abstract
Bartonella quintana, an emerging gram-negative pathogen, may cause trench fever, endocarditis, cerebral abscess and
bacillary angiomatosis usually with the absence of septic shock in humans. B. quintana lipopolysaccharide (LPS), a deep rough
endotoxin with strong reactivity in the limulus amebocyte lysate (LAL)-assay, was studied in human whole blood and in a rat
model. A significant (P< 0.05) increase of interleukin-8 (IL-8) concentration, comparable to the level induced by enterobacterial
LPS, was stimulated in the human whole blood by B. quintana LPS. Isolated human neutrophils delayed their apoptotic behavior
in the presence of B. quintana LPS. In the rat, B. quintana LPS induced a significant (P < 0.001) increase in white blood cell
count, both 30 and 60 min after intravenous injection. Such leukocytosis was inhibited by pretreatment with prazosin, an a-
adrenergic antagonist. B. quintana LPS did not significantly change heart rate (HR), hematocrit (HCT) and platelet count in the
above reported in vivo model, and regarding mean blood pressure (MAP) only a very early (5 min after LPS) and mild (yet
significant) hypotension was observed. In contrast, a long-lasting decrease of MAP was found in Salmonella minnesota R595
LPS-treated animals. Blood TNFa levels did not change significantly from the baseline in rats injected with either saline or with
B. quintana LPS, on the contrary S. minnesota R595 LPS-injected animals showed substantial increase of TNFa levels up to
2924 pg/ml at 60 min after LPS injection. B. quintana LPS as well as Salmonella LPS-injected rats exhibited an increase of the
blood levels of GRO/CINC-1, particularly at 240 min after LPS administration. Apical part of rat gut villi showed several
TUNEL-positive cells in tissue sections from B. quintana LPS-treated animals. Taken together, our data demonstrates that B.
quintana LPS is able to selectively stimulate some inflammatory mediators. B. quintana LPS-induced leukocytosis appears
mediated by an a-adrenergic receptor. The delayed apoptotic process of leukocytes and the chemokine increase may explain the
apoptotic cells found in the rat gut and the inflammatory reactions in some human Bartonella diseases. This peculiar
1567-5769/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S1567-5769(03)00059-6
* Corresponding author.
G. Matera et al. / International Immunopharmacology 3 (2003) 853–864854
inflammatory pattern induced by B. quintana LPS, may partially account for the lack of severe septic shock, observed in human
B. quintana infections.
D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Neutrophils; Endotoxins; Interleukins
1. Introduction tis and in several others disease due to Bartonella)
Together with well-characterized acute and chronic
diseases such as trench fever, bacteremia, endocardi-
tis, bacillary angiomatosis, osteitis, present knowledge
about Bartonella quintana infection includes a broad
range of non-specific clinical symptoms and signs
[1,2]. Cases reported such as suspected bacteremia
due to B. quintana might represent only a small
percentage of the patients actually infected, as sug-
gested by the results of the Seattle seroprevalence
survey [3]. Chronic B. quintana bacteremia associated
with severe headaches and leg pain as well as with
low platelet counts, has been reported very recently in
homeless patients [4]. Ileitis has been associated with
Bartonella infection in humans [5].
Pathogenic mechanisms of Bartonella infections
are poorly understood, particularly in vivo.
Endothelial changes play a crucial role in bacil-
lary angiomatosis and in the heart valves pathology
of patients suffering from B. quintana endocarditis
[6,7].
Also, monocytes, macrophages and neutrophils
might be involved in engulfment of Bartonella spp.
[8].
LPS found in the outer membrane of gram-
negative envelope, may be released as cell wall
blebs [9], and represents one of the bacterial com-
ponents mainly responsible for pathogenic mecha-
nisms [10]. However, very little information is
available on the pathogenic role of Bartonella LPS
[11]. In our laboratory, we have extracted and
characterized B. quintana LPS and we have also
investigated the in vitro features of LPS interaction
with an endothelial cell line, as a major intracellular
replicative site of B. quintana [12]. Up to our
knowledge, it was not addressed the question why
a bacterium, that may produce periodic bacteremias
(e.g. as in trench fever) and that stimulates clinical
features (e.g. again as in trench fever, in endocardi-
quite typical for a conventional endotoxin bearing
organism, does not usually cause septic shock
[1,4,8].
The aim of the present paper is to investigate some
effects of B. quintana LPS after in vivo administra-
tion in the rat and following in vitro stimulation of
the human whole blood and of the isolated human
PMN. Evaluation and neutralization of endotoxic
activity of this LPS using the LAL-assay were also
reported.
2. Materials and methods
2.1. Evaluation and neutralization of endotoxic
activity of B. quintana LPS
The endotoxic activity of extracted B. quintana
LPS was evaluated using the limulus amebocyte
lysate test (LAL test) (QCL-1000; BioWhittaker Bio-
products, Walkerville, MD, USA). As reference com-
pounds for LAL experiments, one rough Re che-
motype LPS (from Salmonella minnesota R595;
Sigma, St. Louis, MO, USA) and one smooth type
LPS (Proteus mirabilis LPS, RIBI, Montana, USA)
were used. Also, its in vitro neutralization by anti-
biotics was evaluated by LAL reactivity inhibition, as
previously reported [13]. Results are shown as O.D. at
405 nm.
2.2. Induction of IL-8 release in human whole blood
after stimulation with B. quintana LPS
Blood samples were obtained from healthy volun-
teers and aliquoted. The first aliquot was added with
0.03 ml of 15% EDTA and tested for differential cell
count using a MAX-M cell counter (Coulter, Luton,
UK). Blood samples exhibited meanF S.E.M. values
for HCT of 46F 1%, for total leukocyte count of
G. Matera et al. / International Immunopharmacology 3 (2003) 853–864 855
7200F 190 cells/Al and for monocyte count of
360F 20 cells/Al. Endotoxin-free heparin (50 IU/ml
final concentration), instead of EDTA, was added to
all the remaining aliquots of each blood sample. A
second aliquot of 1.8 ml of each blood sample
(control) was added with 0.2 ml of sterile saline and
processed immediately as reported below. Remaining
aliquots of each blood sample were distributed into
sterile polypropylene tubes (1.8 ml/tube), and 0.2 ml
volume of purified LPS (LPS-treated; final concen-
tration 1000 ng/ml) or the same volume of sterile
saline (control) was added. Thus, 648� 104 WBC/ml
was used. Whole blood incubation was performed in
non-rotating tubes at 37 jC. After 2, 4 and 24 h of
incubation, plasma was separated from blood cells by
centrifugation for 5 min at 1500� g, and stored at
� 20 jC in different aliquots. IL-8 concentration was
evaluated by enzyme-linked immunosorbent assay
(ELISA).
2.3. Inflammatory mediators detection by ELISA
Human IL-8, as well as rat TNFa and GRO/CINC-
1 concentrations were measured by commercially
available ELISA kits (Amersham Pharmacia Biotech,
Italy). The lower limit of sensitivity was 4 pg/ml for
IL-8 assay. The sensitivity of rat TNFa and GRO/
CINC-1 assays were 10 and 4.7 pg/ml, respectively.
Concerning IL-8 concentration in human whole
blood, data obtained from each assay were corrected
for blood dilution value in order to reflect the IL-8
concentration as pg/ml of undiluted blood. Since
values of HCT and differential WBC showed only
negligible differences among samples from different
donors, we did not try to adjust IL-8 values for
monocyte and/or white cell count.
2.4. Human PMN apoptosis
Neutrophils were purified by dextran sedimentation
from blood samples obtained by human volunteers.
Cells were counted by hemocytometer, adjusted to
5� 105 cells/ml and cultures were carried out into
a 3-cm petri dish, containing a coverslip and 2 ml
of RPMI 1640, with 10% fetal calf serum. Then,
B. quintana LPS or S. minnesota R595 LPS, after
dilution in RPMI 1640, was added to treated cultures
in order to reach a final concentration of 10 ng/ml.
Incubation was carried for different times in a 5% CO2
atmosphere. Before microscopic evaluation of apop-
tosis, the coverslip of the PMN culture to be examined
was picked up with sterile forceps and placed face-up
down on a glass slide, bearing 10 Al of kit buffer plus1Al annexin and 1 Al propidium (Annexin V-EGFP
staining kit, MBL, NaKa-Ku Nagaya, Japan). Appli-
cation of a DNA staining, propidium iodide (PI),
allowed to discriminate apoptosis from necrosis. Cells
in earlier stages of apoptosis, which have a normal
appearance and display a green fluorescence on outer
leaflet, are named annexin-positive cells (A cells).
Cells in later stages of apoptosis begin to lose mem-
brane integrity and show an additional red fluores-
cence on strongly condensed nuclei. These cells are
reported as annexin-propidium-positive cells (AP
cells) and may be easily distinguished from necrotic
cells which appear diffusely stained with PI, and are
reported as propidium-positive cells (P cells), with
nuclei conserving their shapes. Percentages of apop-
totic cells were determined evaluating at least 500 cells
per sample, and counting A, AP and P cells, using a
fluorescence microscope (Leica Microsystem Wetzlar
GmBH, Wetzlar, Germany).
2.5. Rat hemodynamic, hematological and cytokine
measurements
Male Wistar rats (200–250 g b.w., Charles River,
Italy) were anesthetized with sodium pentobarbital
(50 mg/kg; i.p.), and instrumented for measurement
of arterial blood pressure and heart rate. Using a
Gould-Statham P23Db pressure transducer attached
to a PowerLab (AD Instruments) and to a Columbus
Instruments Computer, blood pressure and heart rate
were continuously displayed and recorded. After
completion of surgical procedures, animals were
allowed to stabilize their cardiovascular parameters
for 20 min. Then, baseline hemodynamic values were
recorded, a blood sample was obtained for white
blood cell and platelet counts and for hematocrit
evaluation and an equal volume of sterile saline
was returned to the animal. After 5 min, animals
received LPS from B. quintana (10 mg/kg, i.v.; in 0.2
ml/100 g b.w. of sterile saline) or LPS from S.
minnesota R595 (10 mg/kg, i.v.; in 0.2 ml/100 g
b.w. of sterile saline) or 0.2 ml/100 g b.w. of sterile
saline (controls) as a slow injection over 2 min.
Table 1
Reactivity of LPS from B. quintana, P. mirabilis and S. minnesota
R595 measured by LAL test
LPS O.D. 405 nm
(pg/ml)B. quintana P. mirabilis S. minnesota R595
1.5 N.D. N.D. N.D.
3.1 0.149F 0.058 N.D. N.D.
6.2 0.351F 0.060 N.D. N.D.
G. Matera et al. / International Immunopharmacology 3 (2003) 853–864856
Further, blood samples were obtained 30 and 60 min
after LPS or saline administration, unless otherwise
specified.
Separate groups of anesthetized animals received
either an a-adrenergic antagonist, prazosin (1 mg/kg,
i.v.; prazosin hydrochloride, Sigma), or saline admin-
istered 20 min before the LPS from B. quintana (10
mg/kg, i.v.) or 0.2 ml/100 g b.w. of sterile saline
(controls), in order to evaluate the role of a-adrenergic
mechanism in the leukocytosis induced by B. quin-
tana LPS.
Hematological measurements were carried out by a
automatic blood cell counter (MAX-M, Coulter).
Information on rat TNF a and GRO/CINC-1 blood
levels were obtained from different groups of anes-
thetized animals treated either with one of the above
LPSs (10 mg/kg, i.v.; in 0.2 ml/100 g b.w. of sterile
saline), or with sterile saline (0.2 ml/100 g b.w., i.v.).
2.6. Histology
In a separate group of experiments, the rats (n = 4
per each group) were anesthetized with sodium pen-
tobarbital 50 mg/kg, i.p. and treated with B. quintana
LPS (10 mg/kg, i.v.; in 0.2 ml/100 g b.w. of sterile
saline) or S. minnesota R595 LPS (10 mg/kg, i.v.; in
0.2 ml/100 g b.w. of sterile saline) or 0.2 ml/100 g
b.w. of sterile saline (controls) as a slow injection over
2 min.
After 3 h, the rats were transcardially perfusion-
fixed with 4% buffered formaldehyde pH 7.4 after a
brief rinse with saline and heparin (0.1%) at room
temperature. Gut, lung, liver and kidney were re-
moved, kept in cold fixative for 2 h, and stored in
phosphate-buffered saline (PBS) overnight. Tissues
were dehydrated in ethanol and embedded in paraffin.
Sections (5 Am) were cut in a microtome and stained
with hematoxylin–eosin (H&E) for light microscopic
examination. Terminal deoxynucleotidyltransferase-
mediated dUTP nick end labelling (TUNEL) was
performed according to the producer’s protocol (MK
500; Takara Biomedical Group, Japan).
12.5 0.587F 0.029 0.160F 0.018 0.042F 0.01225 0.694F 0.042 0.245F 0.029 0.268F 0.020
50 1.095F 0.048 0.385F 0.021 0.671F 0.061
100 1.297F 0.050 0.831F 0.018 0.941F 0.031
Data are meansF S.E.M. of five experiments, which were carried
out in duplicate.
N.D.: not detectable.
3. Statistical analysis
The data are reported as meansF standard error of
the mean (S.E.M.), unless otherwise specified. One-
way ANOVA was used to analyse the data and
significant differences between groups were deter-
mined by Fisher’s Protected Least Significant Differ-
ence (PLSD) test. A P < 0.05 was considered to be
statistically significant.
4. Results
4.1. Evaluation and neutralization of endotoxic
activity of B. quintana LPS
Data concerning LAL-test reactivity are reported in
Table 1. When compared to other reference endotox-
ins, B. quintana LPS showed a strong endotoxic
activity, as measured by LAL-test. Furthermore, this
activity was also evident when concentrations of 3.1
and 6.2 pg/ml of endotoxin were tested, while other
reference endotoxins resulted undetectable at these
two low concentrations.
The direct in vitro neutralization of the LPS from
B. quintana by antibiotics was evaluated using the
technique of the inhibition of the reactivity of bacte-
rial LPS in the LAL-test and the data are reported in
Table 2. A significant and dose-dependent reduction
of the LPS reactivity due to polycationic antibiotics
and particularly to polymyxin B can be observed. On
the contrary, no significant modifications of the LPS
reactivity was caused by the beta-lactam used (cefta-
zidime). A comparable reduction of LPS reactivity in
the LAL test was found using one reference endotoxin
(from S. minnesota R595) plus the above polycationic
Table 2
Effect of polymyxin B (PB), tobramycin (TOB), isepamicin (ISE)
and ceftazidime (CTZ) on the reactivity of B. quintana, S.
minnesota R595 and P. mirabilis LPS (Control, 50 pg/ml) measured
by LAL test
Antibiotic O.D. 405 nm
(pg/ml)B. quintana S. minnesota R595 P. mirabilis
Control 1.095F 0.048 0.806F 0.030 0.398F 0.25
PB (10) 0.875F 0.006 0.513F 0.087* 0.391F 0.016
PB (100) 0.560F 0.033* 0.150F 0.067* 0.354F 0.026
PB (1000) 0.381F 0.046* 0.118F 0.001* 0.381F 0.006
TOB (10) 0.789F 0.020 0.554F 0.014* 0.365F 0.007
TOB (100) 0.732F 0.027* 0.322F 0.064* 0.372F 0.001
TOB (1000) 0.697F 0.025* 0.171F 0.001* 0.386F 0.014
ISE (10) 0.841F 0.018 0.327F 0.036* 0.397F 0.018
ISE (100) 0.708F 0.039* 0.178F 0.021* 0.394F 0.013
ISE (1000) 0.650F 0.038* 0.104F 0.019* 0.372F 0.007
CTZ (10) 0.866F 0.003 0.791F 0.053 0.352F 0.026
CTZ (100) 0.784F 0.016 0.890F 0.032 0.374F 0.011
CTZ (1000) 0.789F 0.007 0.861F 0.022 0.380F 0.007
Data are meansF S.E.M. of five experiments, which were carried
out in duplicate. *P< 0.01 vs. antibiotic-free LPS Control, by
Fisher’s PLSD test.
G. Matera et al. / International Immuno
antibiotics. On the contrary, a lack of LPS neutraliza-
tion was observed when we used the well-known
polymyxin-resistant LPS from P. mirabilis.
Fig. 1. Kinetics of the release of IL-8 in human whole blood stimulated w
R595 LPS (1000 ng/ml, i.v.; open circles). Instead of LPS, sterile saline w
meansF S.E.M. of data from three healthy donors tested in duplicate. Each
time, by Fisher’s PLSD test.
4.2. Induction of IL-8 release in human whole blood
after stimulation with B. quintana LPS
The activity of B. quintana LPS on IL-8 release is
shown in Fig. 1. We found a significant (P < 0.05 vs.
control values) increase of IL-8 along with the time
(2–4–24 h, after 1000 ng/ml of LPS). The LPS from
S. minnesota R595 (1000 ng/ml) stimulated a slower,
but significant, increase of IL-8 at 4 h after LPS,
whereas after 24 h both LPS induced almost the
same levels of this chemokine. Lower dosages of
both LPS did not achieve a statistically significant
IL-8 increase in comparison to control (data not
shown).
4.3. Human PMN apoptosis
Human neutrophil apoptosis was studied by An-
nexinV–Propidium staining. PMN were scored as
‘‘A +AP’’ (Annexin plus Annexin/Propidium posi-
tive, that is early apoptotic cells plus late apoptotic
ones) or ‘‘P’’ (Propidium-only—positive, that is nec-
rotic cells).
In control PMN, a rapid increase of A +AP per-
centage was found up to the sixth hour, then the
pharmacology 3 (2003) 853–864 857
ith B. quintana LPS (1000 ng/ml, open triangles), or S. minnesota
as added to control human whole blood (open squares). Values are
experiment was carried out twice. *P < 0.05 vs. control at the same
Fig. 2. Apoptosis induced by B. quintana LPS on PMN evaluated by the Annexin-V Propidium staining. Kinetics of percentages of early + late
apoptotic (A +AP) cells observed in control human PMN (open squares), in B. quintana LPS-stimulated human PMN (open lozenges), or in S.
minnesota R595 LPS-stimulated human PMN (open circles). It is also shown the kinetic of percentages of necrotic (P) cells found in control
human PMN (crossed squares), in B. quintana LPS-stimulated human PMN (crossed lozenges), or in S. minnesota R595 LPS-stimulated human
PMN (crossed circles). Data are shown as mean percentageF S.E.M. of positive cells, out of at least 500 counted ones, during three experiments
carried out in duplicate. See the text for details.
G. Matera et al. / International Immunopharmacology 3 (2003) 853–864858
percentage of A +AP cells decreased to a value lower
than that observed at 2 h; on the contrary, the curve
representing the necrotic PMN rose slowly but con-
stantly and crossed the descending A+AP cell curve
between 6 and 24 h (Fig. 2). Other investigators
reported steadily rising proportions of necrotic PMN
after 18–30 h of in vitro culture. On the contrary, the
percentage of early apoptotic cells that had showed an
Table 3
Changes (%) of mean arterial pressure (MAP) of Wistar rats at 5, 15, 30, 45
mg/kg) and S. minnesota R595 LPS (10 mg/kg), as well as sterile saline
Treatment Time (min)
5 15
B. quintana LPS 75.2F 5.8 ( P= 0.013) 95.6F 5.4
S. minnesota R595 LPS 70.1F 4.8 ( P= 0.006) 76.8F 9.1
Control 103.9F 5.0 92.9F 1.9
Values are meansF S.E.M. of data from six animals.
P vs. control at the same time, by Fisher’s PLSD test.
increase until 18–30 h, then exhibited a progressive
decrease [37].
B. quintana LPS (10 ng/ml) caused a delay of the
apoptotic course of human neutrophils (Fig. 2). The
concentration of 10 ng/ml of LPS was selected during
preliminary experiments, as the lowest dosage of LPS
producing a substantial difference in the apoptosis
percentage when compared to the control PMN. On
and 60 min after intravenous administration of B. quintana LPS (10
(control)
30 45 60
83.9F 4.7 87.8F 7.4 79.4F 6.2
(P= 0.04) 73.6F 8.3 81.6F 17.6 70.5F 17.1
81.9F 5.0 88.5F 13.0 79.8F 8.5
Table 5
Effect of prazosin pretreatment (1 mg/kg, i.v., 20 min before LPS or
saline) on changes (%) of WBC counts of Wistar rats at 30 and 60
min after intravenous administration of B. quintana LPS (10 mg/
kg), or sterile saline
Pretreatment Treatment Time (min)
30 60
Saline B. quintana
LPS
144F 15.4 149.4F 16.7
Prazosin B. quintana
LPS
96.8F 15.1
(P= 0.008)
93.8F 18.4
(P= 0.002)
Prazosin Saline 108F 14.1 104.5F 5.0
Values are meansF S.E.M. of data from six animals.
P vs. saline plus B. quintana LPS-injected rats at the same time, by
Fisher’s PLSD test.
G. Matera et al. / International Immunopharmacology 3 (2003) 853–864 859
the contrary, higher concentrations did not modify the
PMN apoptotic behavior already observed following
10 ng/ml of LPS.
PMN added with B. quintana LPS showed a
slowly increasing A +AP curve with a reduced
slope for all the experimental period, while the P
curve was always close to the baseline values; the
two curves never crossed each other within 24 h.
We observed a similar trend using S. minnesota
R595 LPS, however, the percentage values were
all greater than in B. quintana LPS-treated PMN
and the corresponding curves were shifted to the
left.
Therefore, the time-course of the A+AP and P
curves of the control PMN demonstrate that the
whole process of apoptosis lasted less than 24 h
for a large percentage of PMN. On the contrary, a
delay of apoptotic process appeared in the PMN
treated with both LPS used, since the A +AP curve
and the P curve did not cross each other during the
first 24 h.
4.4. Rat hemodynamic, hematological and cytokine
measurements
B. quintana LPS did not significantly change
heart rate (HR), hematocrit (HCT) and platelet count
in the rat, however, mean arterial pressure (MAP)
values decreased significantly (P= 0.013 vs. control
at same time) at 5 min after LPS injection (Table 3).
As expected the LPS from Salmonella induced a
significant hypotension at 5 and 15 min after LPS
injection.
Salmonella LPS caused a significant thrombocyto-
penia at 30 and 60 min and a significant leukopenia
after 60 min since its administration (Table 4). B.
Table 4
Changes (%) of WBC and platelet counts of Wistar rats at 30 and 60 min af
minnesota R595 LPS (10 mg/kg), as well as sterile saline (control)
Treatment Time (min)
30
WBC Platelets
B. quintana LPS 154.2F 14.5 ( P< 0.001) 105.1F 0.9
S. minnesota R595 LPS 70.4F 9.8 74.2F 5.2 ( P
Control 89.1F 4.6 114.3F 15.3
Values are meansF S.E.M. of data from six animals.
P vs. control at the same time, by Fisher’s PLSD test.
quintana LPS consistently induced a significant in-
crease in white blood cell (WBC) counts after intra-
venous injection in Wistar rats (Table 4). It was noted
that 30 and 60 min after B. quintana LPS, WBC
increased up to 154.2F 14.5% and 158.4F 15.8%
compared with basal level (P < 0.001 vs. control at
the same time).
Using separate groups of animals, we found that in
prazosin-pretreated B. quintana LPS-injected rats,
WBC were found 96.8F 15.1% and 93.8F 18.4%
of the basal level at 30 and 60 min after LPS. On the
contrary, in saline-pretreated B. quintana LPS-injected
animals, WBC changes overlapped those high values
found in unpretreated B. quintana LPS-injected rats.
In prazosin-pretreated LPS vehicle-injected animals,
WBC showed only negligible changes from baseline
at both times (Table 5). Therefore, prazosin blunted
the B. quintana LPS-induced increase of WBC, with-
out producing any WBC change in LPS vehicle-
treated animals.
ter intravenous administration of B. quintana LPS (10 mg/kg) and S.
60
WBC Platelets
158.4F 15.8 ( P < 0.001) 114.1F11.4
< 0.001) 49.2F 6.8 ( P= 0.004) 85.1F 5.2 ( P= 0.04)
82.9F 0.5 104.8F 1.0
Fig. 3. Kinetics of blood levels of TNFa (A) and GRO/CINC-1 (B)
in the rat injected with B. quintana LPS (10 mg/kg, i.v.; open
lozenges), or S. minnesota R595 LPS (10 mg/kg, i.v.; open circles).
Data are meansF S.E.M. from groups of five rats injected with
either LPS (from B. quintana or from S. minnesota R595 LPS) or
with saline (open squares). *P< 0.05 vs. control at the same time,
by Fisher’s PLSD test.
Fig. 4. (A) Apical part of rat gut villi exhibited apoptotic cells
(mainly villi) in B. quintana LPS-injected (10 mg/kg, i.v.) animals,
as shown by TUNEL-processed tissue sections. (B) Animal injected
with LPS from S. minnesota R595 (10 mg/kg, i.v.) showed similar
apoptotic modifications of gut villi. (C) Absence of apoptotic cells
in TUNEL-processed tissue sections obtained from gut of saline-
injected control rats. Representative findings from one of the four
animals used for each treatment.
G. Matera et al. / International Immunopharmacology 3 (2003) 853–864860
Data from experiments carried out in parallel with
different groups of rats injected with either LPS (from
B. quintana or from S. minnesota R595 LPS) or with
saline, showed that blood TNFa levels did not move
from baseline in saline-injected rats. Similarly, in the
rats stimulated by B. quintana LPS, TNFa did not
change from background levels. On the contrary, S.
minnesota R595 LPS-injected animals showed a sharp
increase of TNFa levels (Fig. 3A).
G. Matera et al. / International Immunopharmacology 3 (2003) 853–864 861
Samples from the above animals showed negligible
modifications over the time in the blood levels of
GRO/CINC-1, after saline injection. However, in
samples of B. quintana LPS-stimulated rats we found
an increase of GRO/CINC-1, which achieved a sig-
nificant (P < 0.05) difference vs. control at 60 min and
at 240 min after the challenge. Also, Salmonella LPS-
injected rats exhibited a significant (P < 0.05) GRO/
CINC-1 elevation at the same times after the stimulus
(Fig. 3B).
4.5. Histological studies
Apical part of rat gut (ileum) villi showed apop-
totic cells (mainly villi; very rarely goblet cells) in B.
quintana LPS-injected animals, as shown by TUNEL-
processed tissue sections (Fig. 4A). Animals injected
with LPS from S. minnesota R595 showed similar
apoptotic modifications of gut villi (Fig. 4B). No
Tuneel-positive cells were evident in gut villi of rats
treated with saline (Fig. 4C). Apoptotic features
showed, refer to samples obtained from rats 3 h after
LPS administration (10 mg/kg, i.v.). The morpholog-
ical examination of H&E-stained sections from B.
quintana LPS-treated animals did show the presence
of an infiltrate of neutrophils in the gut villi (data not
shown).
No morphological modifications were seen in lung,
liver and kidney in all the animals examined.
5. Discussion
This is the first report on the effects of B. quintana
LPS on IL-8 in human whole blood and on human
PMN apoptosis. Also, we evaluate previously not
addressed activities of B. quintana LPS on the car-
diovascular system, hematological values and on
tissue apoptosis in the rat. For this purpose we used
B. quintana LPS isolated from aqueous phase follow-
ing Westphal and Jann procedure, as modified by
Minnick [14]. Other investigators reported data on
B. quintana LPS extracted from phenol phase [11].
The two products are quite different and we used the
aqueous phase extract because KDO percentage and
the protein content are more satisfactory. In particular,
KDO amount is comparable with that found in the
enterobacterial LPS and the protein percentage of our
product is similar to that reported by other authors in
water–phenol extracted LPS [15].
In our laboratory, we have previously demonstrated
that lipopolysaccharide extracted from B. quintana
Oklahoma strain showed a molecular weight of about
5000 with a ‘‘deep rough’’ chemotype [12]. This
finding is consistent with results reported by others
dealing with the LPS isolated from Bartonella bacil-
liformis [14].
B. quintana LPS exhibited marked endotoxicity
and was significantly neutralized with polycationic
antibiotics in an LAL-assay model (see Tables 1 and
2). This observation may contribute to explaining in
vitro bactericidal activity of aminoglycosides against
many species of genus Bartonella [16] and the suc-
cess of therapeutic aminoglycoside use in some clin-
ical cases of bartonellosis [8]. The absence of Proteus
LPS neutralization by polycationic antibiotics has
been reported to correlate with the esterification of
the core-lipid A phosphates and core KDO, which has
been found in Proteus LPS [17]. On the contrary, LPS
from B. quintana, as well as from S. minnesota R595
might make such acidic groups (due to the lack of
esterification) available to polycationic antibiotics.
Although our B. quintana LPS showed a strong
endotoxic activity in LAL-assay (where it appeared
more potent than other reference LPS tested), it was
not able to induce a substantial increase of TNFa
levels in human whole blood (data not shown) and in
rats (see Results).
In B. quintana LPS-challenged human endothelial
cells, we previously demonstrated both transcriptional
and protein low levels of TNFa [12]. These data
underlie that the high endotoxic reactivity, showed
by LAL-test, is not associated to a potent proinflam-
matory activity in very different models and this is
particularly true looking at TNFa levels in the rat.
Such discrepancies have been demonstrated for other
LPS [18].
However, in this study we show that IL-8 is
increased in B. quintana LPS-stimulated human
whole blood. In a previous study, IL-8 has been found
to be the major chemokine induced by B. quintana
LPS on endothelium [19]. Moreover, endothelial IL-8
has been demonstrated to be also upregulated at tran-
scriptional level, as indicated by mRNA semiquanti-
tative PCR findings [12]. In addition, IL-8 has been
reported to be a crucial factor in LPS-mediated angio-
G. Matera et al. / International Immunopharmacology 3 (2003) 853–864862
genesis [20] and may contribute to the proliferative
lesions found in both human and animal bartonellosis.
IL-8 level may be even greater than we found, due to
interactions of cytokines and chemokines with gran-
ulocytes, erytrocytes and plasma soluble receptors.
Such interactions may be overcome by using a deter-
gent such as Triton X-100, but such detergent may
interfere [21] with non-electrochemiluminescence
assay of cytokines (e.g. ELISA).
Blood levels of TNFa did not change in our rats
after B. quintana LPS treatment, while reference
Salmonella LPS caused a substantial increase. The
low but detectable levels of TNFa found in baseline
blood samples and in samples from control animals
may be explained with a moderate stress due to the
anesthesia and to the effect of intravascular catheter
used for removal of blood samples [22].
Our data showed that GRO/CINC-1, the rat’s
chemokine equivalent to human IL-8 [23], is en-
hanced by B. quintana LPS treatment. GRO/CINC-1
has been demonstrated to play a potential role in
neutrophil activation during the rat’s response to
various inflammatory stimuli including LPS [23]. It
has been reported that in IL-8-treated primates, as well
as in CINC-administered rats, TNFa levels do not
increase, and that co-injection of LPS and anti-CINC
antibody, causes a significant increase of TNFa in the
rat [24]. Rats challenged with B. quintana LPS
showed only a very early and mild (yet significant)
hypotension. On the contrary, a sharp and long-lasting
decrease of MAP was observed in S. minnesota R595
LPS-treated animals. TNFa is well known for its
direct hypotensive effect, which may take place within
minutes to hours, both in human [25] and in the rat
[26]. On the other hand, the CXC chemokine IL-8 has
been reported to exhibit beneficial antishock effects
[27].
Thus, the lack of TNFa increase and the beneficial
cardiovascular effects of the rat CXC chemokine,
GRO/CINC-1 might explain the lack of a long-lasting
hypotension in B. quintana LPS-treated rats. How-
ever, the very early effect on the MAP, produced by
both LPS used may suggest a mediator, different from
TNFa [26], rapidly released after LPS and with a very
quick time course. Other authors demonstrated that
the maximal hypotensive response was observed at 10
min after LPS injection in anesthetized rats. Such
early reduction in blood pressure was associated with
enhanced plasma concentration of norepinephrine and
cardiac functional alterations [28]. It has already been
reported that pretreatment of LPS-injected rats with a-
adrenergic antagonist ameliorated survival and shock
sequelae [29]. In preliminary experiments, we found
that rat pretreatment with the a-adrenergic antagonist
prazosin abolished the very early hypotensive effect
of Salmonella and Bartonella LPSs (data not shown).
Overall, an a-adrenergic mechanism may play a role
in the early hypotension caused by both Bartonella
and Salmonella LPSs.
Moreover, in the present study we demonstrate a
consistent and significant increase of PMN in blood
samples of rats injected with B. quintana LPS. Hyper-
leukocytosis has also been observed during human B.
quintana infection [8]. Altenburg et al. [30] reported
that LPS-stimulated neutrophilia in the rat is mediated
by an a1-adrenoceptor. Our data on prazosin-mediated
inhibition of B. quintana LPS -induced PMN change,
suggest that such an event is probably mediated by the
catecholamine norepinephrine through an a-adrener-
gic receptor activation. Quick neutrophilia by cate-
cholamine-mediated demargination might fit well
with early neutrophilia observed after B. quintana
LPS. On the other hand, TNFa, a major cytokine
inducing neutropenia by PMN margination [31], did
not appear stimulated by B. quintana LPS in this
study and was found to be downregulated by the same
LPS in a previous investigation [12].
To the extent of our knowledge, no data are
available from the literature on chemokine levels
during in vitro or in vivo experiments using B.
quintana products and in particular LPS. Regarding
TNFa it has been demonstrated that this cytokine was
released by J774, a murine macrophage cell line, after
in vitro stimulation with heat-killed Bartonella hense-
lae [32].
The pathological study of tissues from rats treated
with B. quintana LPS also demonstrated that such
LPS is able to induce apoptosis when injected in rats
and that gut villi show a strong sensitivity to endo-
toxin-induced cellular damage. This is probably due,
even in absence of a substantial hypotension, to a
damage caused by chemokines-stimulated leukocytes.
This hypothesis is consistent with the presence,
reported in this study, of neutrophil infiltration at the
gut villi level in treated-LPS, but not in untreated
animals.
G. Matera et al. / International Immunopharmacology 3 (2003) 853–864 863
Despite the fact that evidence of a modulatory
effect of LPS on cellular apoptosis in vivo and in
vitro abound, many investigators report divergent
results. Even among phagocytes, LPS has been dem-
onstrated to increase macrophage apoptosis and delay
neutrophil death [33].
Our data are consistent with a positive influence of
LPS on rat villi cells apoptosis, that is apparently in
contrast with the delay observed in the neutrophil
apoptosis. However, the ‘‘sensitivity’’ of the gut to
LPS could be explained with the experimental obser-
vation of Schmidt et al. [34] that a reduction of villus
blood flow, due to a vasoconstriction in the central
villus arterioles, occurs during normotensive endotox-
emia in LPS-treated rats. Recently, ileitis has been
associated with Bartonella infection in humans [5].
Moreover, we observed an increased lifespan of
LPS-stimulated human PMN; this delay of PMN
apoptosis is consistent with previous reports evaluat-
ing the same parameter after neutrophil stimulation
with conventional LPS from enterobacteria, as well as
with the much less potent LPS from Helicobacter,
Porphyromonas and Bacteroides [35,36]. Bacteria
bearing a low-potency LPS may take advantage of a
lack of a very effective inflammatory and immune
response. Therefore, they can maintain a fair inflam-
matory activity for a much longer time, than bacteria
with conventional LPS, because the LPS tolerance is
substantially absent, thus leading to a chronic inflam-
mation [35,36].
In conclusion, our results (obtained in two different
models) are evidence that B. quintana LPS is able to
act selectively on certain inflammatory mediators (e.g.
chemokines) and this could be a very important
pathogenic mechanism in bartonellosis. Indeed, the
weak stimulus for TNFa release induced by B. quin-
tana LPS in comparison to conventional LPS from
enterobacteria may account for the lack of acute
inflammatory response and the absence of long-last-
ing hypotension evident both in our experimental
model and in clinical reports of bartonellosis. This
pathogenetic feature may favor a more chronic evo-
lution of the Bartonella-caused diseases probably due
to the lack of quick clearance of the bacteria; such a
picture has been proposed [35,36] for other long-
lasting bacterial diseases associated with low TNFa
levels, high IL-8 release and a delay of apoptosis of
different cell types including neutrophils.
Acknowledgements
We are indebted to Dr. D. Raoult, University of
Marseille, for the kind gift of B. quintana, Oklahoma
strain, used in present work. Our thanks go to Mr.
Giovanni Politi, D. Saturnino, A. Macri and S. Frustaci
for excellent technical support.
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