# Chronic Brucellosis Patients Retain Low Frequency of  CD4+ T-Lymphocytes Expressing CD25 and CD28 afterEscherichiacoli LPS Stimulation of PHA-Cultured PBMCs

Authors: Panagiotis Skendros; Alexandros Sarantopoulos; Konstantinos Tselios; Panagiota Boura
Journal: Clinical and Developmental Immunology
 (2008)

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## Abstract

Chronic brucellosis patients display a defective Th1 response to PHA. We have previously shown that heat-killed B. abortus (HKBA) can downregulate the PHA-induced increase of CD4+/CD25+ and CD14+/CD80+ cells of brucellosis patients. In the present study, we investigate the effect ofE. coli LPS, as a potent stimulant of monocytes and autologous T-lymphocytes, on the PHA-cultured PBMCs of the same groups of patients. Thirteen acute brucellosis (AB) patients, 22 chronic brucellosis (CB) patients, 11 “cured” subjects, and 15 healthy volunteers were studied. The percentage of CD4+/CD25+ and CD4+/CD28+ T-lymphocytes as well as CD14+/CD80+ monocytes were analyzed by flow cytometry after PBMCs culture with PHA plus E. coli LPS. A significant decrease in the percentage of CD4+/CD25+ and CD4+/CD28+ T-lymphocytes was observed in CB compared to AB. In HKBA cultures, compared to E. coli LPS-cultures, there was a significant reduction of CD4+/CD25+ T-lymphocytes in all groups and CD14+/CD80+ in patients groups. We suggest that Brucella can modulate host immune response, leading to T-cell  anergy and chronic infection.


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## Body

## 1. Introduction

Brucellosis is the most
common zoonotic disease with worldwide distribution. More than 500 000 new
cases are reported annually [1, 2]. It is caused by intracellular pathogens of
the genus  Brucella that have their natural reservoir in domestic and wild animals
[1]. The disease is transmitted to humans by consumption of contaminated dairy
products or by occupational contact with infected animals. In addition, Brucella species are thought to be a biowarfare
agent and recently reported to be a significant cause of travel and
immigration-related infection [1, 3–5].In Greece, as in other Mediterranean
countries, brucellosis remains a major disease of economic and human health
importance despite efforts for animal control and eradication projects [2, 4]. Besides minimal brucellosis mortality in
humans, the disease causes high clinical morbidity, protean clinical
manifestations, and complications, as any organ can be affected [1, 6, 7].
Despite early diagnosis and treatment, approximately 10–30% of patients
develop chronic persistence disease which is characterized by atypical clinical
picture, chronic fatigue syndrome, and relapses [4, 6–8].Host protection against Brucella spp. depends on
cell-mediated immunity, involving mainly activated macrophages, dendritic cells,
and T-cells [9–13]. T-helper 1 (Th1) immune response is essential
for the clearance of Brucella infection and Brucella antigens induce the production of Th1 cytokines in
humans [9–11, 14]. On the other hand, Brucella can survive and replicate in host macrophages and
dendritic cells subverting innate immunity and evading adaptive immune
mechanisms [8, 12, 13, 15, 16]. Recent data report that human dendritic cells infected by living Brucellae are poor
inducers of human naïve T-lymphocyte proliferation and present low production
of interleukin
(IL)-12 which is essential to drive a Th1 immune response [13, 16]. Diminished production
of Th1 cytokines (IFNγ and
IL-2) has been
associated with T-cell unresponsiveness (anergy) to Brucella antigens
and disease chronicity [17–19]. In addition, proliferation response of
CD4+ T-lymphocytes to phytohaemagglutinin (PHA) in chronic brucellosis patients
was found to be significantly low [20]. Recently,
we have shown low frequency of ex vivo
and PHA-cultured CD4+ T-lymphocytes expressing IL-2 receptor alpha
(CD25) in chronic brucellosis patients [21].CD80/CD28 costimulation
enhances the interaction of antigen/major histocompatibility complex (MHC) with
T-cell receptor (TCR) and is critical for an adequate induction and maintenance
of the Th1 response [22]. When we
studied CD80/CD28 costimulation in human brucellosis, it was indicated
that heat-killed B.
abortus (HKBA) significantly downregulated the PHA-induced increase of
CD80+ monocytes [23]. This
effect could be attributed to Brucella lipopolysaccharide (LPS) which is thought to be one of the virulence
factors that endow the pathogen with
the ability to escape the immune [8, 15, 24]. Brucella smooth LPS is considered
to be very important for bacterium survival and replication in the host [24–26]. It is an unconventional, nonendotoxic LPS, as compared with classical LPS
from enterobacteria such as E. coli [27]. Enterobacteria LPS is a potent stimulant of monocytes for the production
of proinflammatory cytokines (such as tumor necrosis factor (TNF)α, IL-1β) and the expression of CD80 [28]. Moreover, it induces the IFNγ secretion, as well as, the upregulation
of CD25 and CD28 by autologous T-lymphocytes [29–32].Thus,
we decided to investigate the influence ofE. coli LPS on CD25
expression and CD80/CD28 costimulation of PHA-stimulated peripheral
blood mononuclear cells (PBMCs) from brucellosis patients and compare the findings with our previous
results regarding the HKBA stimulation of PBMCs in the same groups of patients
[23].


## 2. Materials and Methods

### 2.1. Subjects

Sixty
one unrelated subjects were included in the study: (a) 35 brucellosis patients;
(b) 11 subjects who had a previous history of brucellosis and were cured at
least 2 years ago; (c) 15 healthy age- and gender-matched volunteers who were
used as controls. Patients were divided in acute (AB) and chronic (CB)
brucellosis groups according to disease history, clinical picture, and
laboratory findings [21, 23].The
AB group included 13 consecutive patients (Table1). All AB patients had a disease
duration ≤8 weeks (mean  ±  SD, 3.9±2.6 weeks). The
diagnosis was based on a compatible clinical picture (Table 2) in combination
with high serum titres of antibrucellar antibodies or a fourfold increase of
the initial titres in two-paired
samples drawn 2 weeks apart. In addition, in 6 out of 13 AB patients, Brucella
melitensis was isolated in blood culture. In 2 patients, brucellar DNA was
detected by PCR analysis in the blood and the serum.Table 1
Demographic data of the groups (AB, CB, “cured,” controls) and CB
subgroups (CB1, CB2) studied.
ABCBCB1CB2CuredControlsn132212101115Female521144Male820119711Age (mean  ±  SD, years)44.7±21.448.1±15.645.3±14.851.6±16.650.3±16.743.8±16.3Table 2
Clinical characteristics of the patients studied.
SymptomsAB (n=13)CB1 (n=12)CB2 (n=10)Fever133Sweating1121Chills91Malaise/fatigue889Arthalgias651Lumbar pain431Headache4Myalgias23Depression1Focal diseaseSpondylitis11Sacroiliitis1Epididymoorchitis1Meningoencephalitis1The
CB group comprised 22 patients (Table1). All CB patients had a disease
duration ≥12 months. Twelve patients were suffering from
the relapsing type of chronic disease (CB1 subgroup, mean disease duration
36.3±18.8 months) which is characterized by periodic attacks of fever, chills,
and arthalgias/myalgias [6, 7, 21, 23] (mean number of relapses 2.3±0.7). At
the time of study entry, CB1 patients were under relapse in accordance to the
clinical picture and the high titres of antibrucellar antibodies. Nine of 12
CB1 patients had also positive PCR analysis in the blood. The variable type of
the chronic disease was presented by 10 of the 22 chronic brucellosis patients
(CB2 subgroup, mean disease duration 32.4±15.9 months). CB2 subgroup patients
experienced no relapses but displayed atypical symptoms (Table 2) and
persistently high serum antibrucellar antibody titres [6, 7, 21, 23].High
antibrucellar antibody titres were considered for the Wright agglutination test ≥1 : 320, for the Coombs agglutination test ≥1 : 320, and for the complement fixation test ≥1 : 32.“Cured”
from brucellosis subjects (Table1) remained symptomless and with negative antibrucellar
antibodies for at least 2 years. Healthy volunteers (Table 1) were tested
serologically for brucellosis and found to be negative.All
CB patients and “cured” subjects had received initial antibiotic treatment
including mainly streptomycin 1gr/day combined with rifampicin 600 mg/day plus
doxycycline 200 mg/day for 2 weeks and rifampicin 600 mg/day plus doxycycline
200 mg/day for 6 to 10 weeks thereafter. In 8 of 22 CB patients and 3 of 11 “cured”
individuals, streptomycin was not administered. In a CB1 patient who suffered
from spondylitis (Table2) treatment lasted for 6 months. In addition, most of
CB patients had received more than one antibiotic course and alternative
therapeutic approaches were used such as cotrimoxazole or ciprofloxacin in
combination with doxycycline and/or
rifampicin. Patients with inadequate treatment were not included in the study.The
exclusion criteria of the study were(i)
coexistence of other infectious, neoplasmatic, or
autoimmune disease;(ii)
administration of antibiotic or
immunostimulating agents for at least 30 days before entering the study;(iii)
recent (≤6 months) vaccination;(iv)
pregnancy.
Informed consent was obtained from all subjects enrolled in the study.


### 2.2. Cell Cultures

Twenty mL heparinized venous blood was collected in sterile tubes and PBMCs were
isolated using density gradient centrifugation (Histopaque-1007, Sigma
Laboratories, St. Louis, Mo, USA). The viability of PBMCs was determined to be
greater than 95%, as indicated by Trypan blue dye exclusion (Sigma Laboratories,
St Louis, Mo, USA).We used
LPS fromE. coli serotype
0111 : B4 (Sigma Laboratories, St. Louis, Mo, USA). This serotype does not share
high antigenic similarity with brucella species [33].Preliminary
experiments in PBMCs from healthy volunteers were carried out to standardize the time and
dose of PHA andE. coli LPS needed for the optimal expression of CD25,
CD80, and CD28. In accordance with previous reports, PBMCs were stimulated with
1, 3, and 10 μg E. coli LPS per
mL of culture medium
[34–36] and no differences were noticed in the expression of the CD80
molecule. Finally, we decided to use the 3 μg/mL concentration, as it has
already been used in another brucellosis study on human monocytes [34].PBMCs
were cultured in triplicate in the presence of 5μg/mL PHA plus 3 μg/mL E. coli LPS
in 24-well plates (Costar, Boston, Mass, USA). PBMCs were 1  ×  106 cells per well of culture plate. Each well contained 1 mL of culture
suspension. Culture medium consisted of RPMI-1640 (Gibco Laboratories, Paisley,
UK) supplemented with 10% fetal calf serum (Gibco Laboratories, Paisley, UK), 2 mmol/l l-glutamine (Sigma Laboratories, St. Louis, Mo, USA),
100 IU/mL penicillin (Sigma Laboratories, St. Louis, Mo, USA),
and 100 mg/mL streptomycin (Sigma Laboratories, St. Louis, Mo, USA).
The cultures were kept at 37°C in a humidified 5% CO2 atmosphere for
48 hours. Approximately 3  ×  106 cells per mL of sample were pelleted in a
round-bottomed centrifuge tube and washed in RPMI-1640. The pellet was
resuspended and 100 mL (~3  ×  105 PBMCs) was stained immediately with
20 μL of the appropriate
monoclonal antibody (MAB).
After 15 minutes incubation at room temperature, the PBMCs were fixed by using
Immunoprep/Q-prep protocol (Coulter, Hialeah, Fla, USA).The
dual staining method (synchronous two-color fluorescence analysis) was used. The MABs included fluorescein
isothiocyanate (FITC)-conjugated anti-CD4 (clone 13B8.2) and anti-CD80 (clone
MAB104) as well as phycoerythrin (PE)-conjugated anti-CD25 (clone B1.49.9),
anti-CD28 (clone CD28.2), and anti-CD14 (clone RMO52). Appropriate isotype
controls included FITC and PE conjugates of IgG1 (clone 679.1Mc7) and IgG1
(clone 679.1Mc7) mouse MABs which were used to assess the nonspecific binding
and to set the threshold between positively and negatively stained cell
populations. All MABs were obtained from Immunotech (Marseille, France).


### 2.3. Flow Cytometry

The
following parameters were analyzed
by flow cytometry after PBMCs stimulation with PHA plus E. coli LPS:(i)
the percentage of CD4/CD25 and CD4/CD28 double-positive T-lymphocytes;(ii)
the percentage of monocytes (CD14) expressing CD80.
PBMCs suspension was used rather than purified
monocytes because we intended to investigate the impact of Brucella infection on monocyte–T-cell
interaction (costimulation from both sides of cell interaction) [23].For
the flow cytometric analysis, EPICS XL (Coulter Electronics, Hialeah, Fla, USA)
was used. Lymphocytes were gated using forward scatter (FS) versus side scatter (SS) dot plots.
CD45 FITC staining (clone ALB12, Immunotech, Marseille, France) was used to
confirm the purity of the lymphocyte population (>97%). Monocytes were
characterized and gated both in an FS versus
SS dot plot and in a dot plot with SS versus PE-labeled CD14 antibody (purity >90%). At least 5000
lymphocytes and 2000 monocytes were acquired and analyzed for their
fluorescence properties. The percentages of surface antigen-stained positive
cells were calculated as the percentage of cells that stained above the
fluorescence value obtained by isotype control antibodies. Only CD14 strongly
positive monocytes were analyzed with respect to percentage expression of CD80
FITC. Results of the mean fluorescence intensity did not provide any additional
information, and are therefore not reported.


### 2.4. Statistical Analysis

Parametric
statistical tests were applied as the variables were distributed normally
(Kolmogorov-Smirnov test). Data were analyzed by Student’st-test or paired t-test (for paired data)
using SPSS software
(SPSS Inc., Chicago, Ill, USA) and were represented as mean  ±  SD.
P-values  <  .05 were considered to be statistically significant.


## 2.1. Subjects

Sixty
one unrelated subjects were included in the study: (a) 35 brucellosis patients;
(b) 11 subjects who had a previous history of brucellosis and were cured at
least 2 years ago; (c) 15 healthy age- and gender-matched volunteers who were
used as controls. Patients were divided in acute (AB) and chronic (CB)
brucellosis groups according to disease history, clinical picture, and
laboratory findings [21, 23].The
AB group included 13 consecutive patients (Table1). All AB patients had a disease
duration ≤8 weeks (mean  ±  SD, 3.9±2.6 weeks). The
diagnosis was based on a compatible clinical picture (Table 2) in combination
with high serum titres of antibrucellar antibodies or a fourfold increase of
the initial titres in two-paired
samples drawn 2 weeks apart. In addition, in 6 out of 13 AB patients, Brucella
melitensis was isolated in blood culture. In 2 patients, brucellar DNA was
detected by PCR analysis in the blood and the serum.Table 1
Demographic data of the groups (AB, CB, “cured,” controls) and CB
subgroups (CB1, CB2) studied.
ABCBCB1CB2CuredControlsn132212101115Female521144Male820119711Age (mean  ±  SD, years)44.7±21.448.1±15.645.3±14.851.6±16.650.3±16.743.8±16.3Table 2
Clinical characteristics of the patients studied.
SymptomsAB (n=13)CB1 (n=12)CB2 (n=10)Fever133Sweating1121Chills91Malaise/fatigue889Arthalgias651Lumbar pain431Headache4Myalgias23Depression1Focal diseaseSpondylitis11Sacroiliitis1Epididymoorchitis1Meningoencephalitis1The
CB group comprised 22 patients (Table1). All CB patients had a disease
duration ≥12 months. Twelve patients were suffering from
the relapsing type of chronic disease (CB1 subgroup, mean disease duration
36.3±18.8 months) which is characterized by periodic attacks of fever, chills,
and arthalgias/myalgias [6, 7, 21, 23] (mean number of relapses 2.3±0.7). At
the time of study entry, CB1 patients were under relapse in accordance to the
clinical picture and the high titres of antibrucellar antibodies. Nine of 12
CB1 patients had also positive PCR analysis in the blood. The variable type of
the chronic disease was presented by 10 of the 22 chronic brucellosis patients
(CB2 subgroup, mean disease duration 32.4±15.9 months). CB2 subgroup patients
experienced no relapses but displayed atypical symptoms (Table 2) and
persistently high serum antibrucellar antibody titres [6, 7, 21, 23].High
antibrucellar antibody titres were considered for the Wright agglutination test ≥1 : 320, for the Coombs agglutination test ≥1 : 320, and for the complement fixation test ≥1 : 32.“Cured”
from brucellosis subjects (Table1) remained symptomless and with negative antibrucellar
antibodies for at least 2 years. Healthy volunteers (Table 1) were tested
serologically for brucellosis and found to be negative.All
CB patients and “cured” subjects had received initial antibiotic treatment
including mainly streptomycin 1gr/day combined with rifampicin 600 mg/day plus
doxycycline 200 mg/day for 2 weeks and rifampicin 600 mg/day plus doxycycline
200 mg/day for 6 to 10 weeks thereafter. In 8 of 22 CB patients and 3 of 11 “cured”
individuals, streptomycin was not administered. In a CB1 patient who suffered
from spondylitis (Table2) treatment lasted for 6 months. In addition, most of
CB patients had received more than one antibiotic course and alternative
therapeutic approaches were used such as cotrimoxazole or ciprofloxacin in
combination with doxycycline and/or
rifampicin. Patients with inadequate treatment were not included in the study.The
exclusion criteria of the study were(i)
coexistence of other infectious, neoplasmatic, or
autoimmune disease;(ii)
administration of antibiotic or
immunostimulating agents for at least 30 days before entering the study;(iii)
recent (≤6 months) vaccination;(iv)
pregnancy.
Informed consent was obtained from all subjects enrolled in the study.


## 2.2. Cell Cultures

Twenty mL heparinized venous blood was collected in sterile tubes and PBMCs were
isolated using density gradient centrifugation (Histopaque-1007, Sigma
Laboratories, St. Louis, Mo, USA). The viability of PBMCs was determined to be
greater than 95%, as indicated by Trypan blue dye exclusion (Sigma Laboratories,
St Louis, Mo, USA).We used
LPS fromE. coli serotype
0111 : B4 (Sigma Laboratories, St. Louis, Mo, USA). This serotype does not share
high antigenic similarity with brucella species [33].Preliminary
experiments in PBMCs from healthy volunteers were carried out to standardize the time and
dose of PHA andE. coli LPS needed for the optimal expression of CD25,
CD80, and CD28. In accordance with previous reports, PBMCs were stimulated with
1, 3, and 10 μg E. coli LPS per
mL of culture medium
[34–36] and no differences were noticed in the expression of the CD80
molecule. Finally, we decided to use the 3 μg/mL concentration, as it has
already been used in another brucellosis study on human monocytes [34].PBMCs
were cultured in triplicate in the presence of 5μg/mL PHA plus 3 μg/mL E. coli LPS
in 24-well plates (Costar, Boston, Mass, USA). PBMCs were 1  ×  106 cells per well of culture plate. Each well contained 1 mL of culture
suspension. Culture medium consisted of RPMI-1640 (Gibco Laboratories, Paisley,
UK) supplemented with 10% fetal calf serum (Gibco Laboratories, Paisley, UK), 2 mmol/l l-glutamine (Sigma Laboratories, St. Louis, Mo, USA),
100 IU/mL penicillin (Sigma Laboratories, St. Louis, Mo, USA),
and 100 mg/mL streptomycin (Sigma Laboratories, St. Louis, Mo, USA).
The cultures were kept at 37°C in a humidified 5% CO2 atmosphere for
48 hours. Approximately 3  ×  106 cells per mL of sample were pelleted in a
round-bottomed centrifuge tube and washed in RPMI-1640. The pellet was
resuspended and 100 mL (~3  ×  105 PBMCs) was stained immediately with
20 μL of the appropriate
monoclonal antibody (MAB).
After 15 minutes incubation at room temperature, the PBMCs were fixed by using
Immunoprep/Q-prep protocol (Coulter, Hialeah, Fla, USA).The
dual staining method (synchronous two-color fluorescence analysis) was used. The MABs included fluorescein
isothiocyanate (FITC)-conjugated anti-CD4 (clone 13B8.2) and anti-CD80 (clone
MAB104) as well as phycoerythrin (PE)-conjugated anti-CD25 (clone B1.49.9),
anti-CD28 (clone CD28.2), and anti-CD14 (clone RMO52). Appropriate isotype
controls included FITC and PE conjugates of IgG1 (clone 679.1Mc7) and IgG1
(clone 679.1Mc7) mouse MABs which were used to assess the nonspecific binding
and to set the threshold between positively and negatively stained cell
populations. All MABs were obtained from Immunotech (Marseille, France).


## 2.3. Flow Cytometry

The
following parameters were analyzed
by flow cytometry after PBMCs stimulation with PHA plus E. coli LPS:(i)
the percentage of CD4/CD25 and CD4/CD28 double-positive T-lymphocytes;(ii)
the percentage of monocytes (CD14) expressing CD80.
PBMCs suspension was used rather than purified
monocytes because we intended to investigate the impact of Brucella infection on monocyte–T-cell
interaction (costimulation from both sides of cell interaction) [23].For
the flow cytometric analysis, EPICS XL (Coulter Electronics, Hialeah, Fla, USA)
was used. Lymphocytes were gated using forward scatter (FS) versus side scatter (SS) dot plots.
CD45 FITC staining (clone ALB12, Immunotech, Marseille, France) was used to
confirm the purity of the lymphocyte population (>97%). Monocytes were
characterized and gated both in an FS versus
SS dot plot and in a dot plot with SS versus PE-labeled CD14 antibody (purity >90%). At least 5000
lymphocytes and 2000 monocytes were acquired and analyzed for their
fluorescence properties. The percentages of surface antigen-stained positive
cells were calculated as the percentage of cells that stained above the
fluorescence value obtained by isotype control antibodies. Only CD14 strongly
positive monocytes were analyzed with respect to percentage expression of CD80
FITC. Results of the mean fluorescence intensity did not provide any additional
information, and are therefore not reported.


## 2.4. Statistical Analysis

Parametric
statistical tests were applied as the variables were distributed normally
(Kolmogorov-Smirnov test). Data were analyzed by Student’st-test or paired t-test (for paired data)
using SPSS software
(SPSS Inc., Chicago, Ill, USA) and were represented as mean  ±  SD.
P-values  <  .05 were considered to be statistically significant.


## 3. Results

There
was a significant increase in the percentage of CD4+/CD25+ T-lymphocytes in AB
patients compared to controls (P=.027).
On the other hand, a significant decrease in the percentage of CD4+/CD25+ T-lymphocytes
was observed in CB group of patients compared to AB (P  =  .006) (Table 3(a)).Table 3
Percentage of CD4+/CD25+,
CD4+/CD28+ lymphocytes, and CD80+ cells of CD14+ population, in PHA-cultures with (a)E. coli LPS and (b) HKBA (results are presented as mean  ±  SD). NS: nonsignificant.
(a)E. coli LPS culturesn%CD4+/CD25+%CD4+/CD28+%CD14+/CD80+AB1337.2±11.643.4±11.347.9±19.9CB2227.9±6.935.7±8.045.9±23.2Cured1130.8±12.839.8±17.221.8±14.4Controls1527.4±10.536.5±14.920.9±12.5P (AB versus cured)  =  NSP (AB versus cured)  =  NSP(AB versus cured)  =  .002P (AB versus controls)  =  .027P (AB versus controls)  =  NSP (AB versus controls)  <  .001P (CB versus cured)  =  NSP (CB versus cured)  =  NSP (CB versus cured)  =  .001P (CB versus controls)  =  NSP (CB versus controls)  =  NSP (CB versus controls)  <  .001P (AB versus CB)  =  .006P (AB versus CB)  =  .024P (AB versus CB)  =  NSCB11226.3±5.934.5±8.749.9±26.5CB21029.9±7.937.1±7.241.1±18.7P (CB1 versus AB)  =  .008P (CB1 versus AB)  =  .039P (CB1 versus CB2)  =  NSP (CB1 versus controls)  =  .003P (CB2 versus controls)  =  .004(b)HKBA culturesAB1322.4±11.535.9±14.530.0±20.8CB2224.8±6.538.8±11.236.5±25.5Cured1124.8±14.838.7±17.920.9±16.7Controls1520.6±10.335.6±16.916.3±13.6NSNSP (AB versus controls)  =  .047P (CB versus controls)  =  .004CB11224.9±7.638.3±13.346.6±26.6CB21024.7±5.139.3±8.724.4±18.7NSNSP (CB1 versus CB2)  =  .038P (CB1 versus cured)  =  .012P (CB1 versus controls)  =  .003Concerning
CD4+/CD28+ T-lymphocytes, no differences were found between AB patients and “cured”
subjects or controls. Similarly, CD4+/CD28+ T-lymphocytes in CB patients did
not significantly differ in comparison to nonpatients groups. When CB group of patients
compared to AB, a significant decrease in the percentage of CD4+/CD28+ T-lymphocytes of CB was observed (P  =  .024) (Table 3(a)).T-lymphocytes
subsets did not significantly differ between the CB1 and CB2 subgroups, however
the diminished percentage of CD4+/CD25+ T-lymphocytes in CB patients was
related to the CB1 subgroup (P  =  .008). In the same manner, CB1 patients
showed significantly lower frequency of CD4+/CD28+ T-lymphocytes (P  =  .039)
(Table 3(a)).Regarding the frequency of CD14+
monocytes expressing CD80 costimulation molecule, a significant increase was
found in both of brucellosis groups in comparison to “cured” (P  =  .002 and .001,
resp.) and controls (P  <  .001).No
differences of the percentage of CD80+ monocytes were observed between the CB1
and CB2 subgroups. However, there was a significant increase in the percentage
of this cellular population between both subgroups of CB patients and controls
(P  =  .003 and .004, resp.) (Table 3(a)).We
compared the above results with the findings yielded from our previous studies
(on the same cultured PBMCs stimulated simultaneously with PHA alone or PHA
plus HKBA, Figure1) [21, 23]. The results between PHA alone and PHA plus E.
coli LPS-cultured PBMCs were similar. On the contrary, when HKBA-cultures,
compared to E. coli LPS-cultures, there was a significant reduction of CD4+/CD25+ T-lymphocytes in
all groups (AB P  =  .005, CB P  =  .042, “cured” P  =  .029, controls P  =  .007) and CD14+/CD80+ monocytes in patients groups (AB P  <  .001,
CB P  =  .05) (Figure 1).Comparison of the PHA-cultures. HKBA addition
downregulates (a) the percentages of CD4+/CD25+
T-cells in all groups and (b) CD14+/CD80+ monocytes in brucellosis patients. Data represent mean values+SD (SE).#P  ≤  .05;
PHA versus PHA  +  E. coli LPS, P⋀  ≤  .05;
P⋀⋀  <  .001; PHA versus PHA  +  HKBA,*P  ≤  .05; **P  <  .001; PHA  +  E. coli LPS versus PHA  +  HKBA.
(a)(b)


## 4. Discussion

Brucella acquires the ability to
establish chronic infection and has evolved strategies to actively modulate the
host immune response [8, 10, 13, 15, 16]. Chronic brucellosis patients display
a defective Th1 response to PHA characterized by low-proliferation response of
CD4+ T-lymphocytes, diminished production of IL-2 and IFNγ, and low frequency of CD4+/CD25+ T-cells
compared to acute brucellosis patients [17–21]. In this study, we
investigated the effect of a potent monocytic stimulant (E. coli LPS) on
the PHA-cultured PBMCs of patients with different clinical forms of
brucellosis, especially focusing on chronic disease.E.
coli LPS stimulation of PHA-cultured PBMCs yielded a significant increase
in the percentage of CD4+/CD25+ T-lymphocytes in AB patients as compared to
controls. This finding comes in
agreement with previous data suggesting increased levels of soluble CD25 and upregulation
of CD4+/CD25+ T-cells in acutely ill brucellosis patients [21, 37, 38].
Generally, previous in vitro and in vivo human studies showed that LPS
stimulation of normal T-lymphocytes upregulates the CD25 expression and the
secretion of IL-2 and IFNγ by
them. Thus, LPS is thought to be a potent inducer of human T-cell proliferation
and Th1 cytokine production [29–32]. However, increased
percentage of CD4+/CD25+ cells in AB compared to CB is rather LPS independent, since,
as we have shown previously, PHA-alone cultures displayed similar result [21] (Figure 1). The higher
percentage of CD4+CD25+ T-cells in
acute brucellosis could be attributed
to the abundant milieu of proinflammatory cytokines observed in the
acute disease [37, 39, 40].On
the other hand, the low frequency of CD4+/CD25+ T-lymphocytes in CB compared to
AB group could be explained as an effect of chronic disease. Chronic brucella
infection could lead to a defective in vitro blastogenesis of CD4+ T-lymphocytes
due to low-proliferation response, reduced production of IFNγ and IL-2, and switching toward Th2
response characterized by an increased percentage of CD3+/IL-13+ T-lymphocytes [17–20]. It had been
suggested that IL-13 can downregulate macrophage functions, such as the
production of IL-12 and the expression of inducible nitric oxide synthase in
response to LPS [19, 41].The
reduction of CD4+/CD25+ T-cells percentage after PHA plusE. coli cultures
was more pronounced in chronic relapsing brucellosis patients (CB1 subgroup),
suggesting that relapses in brucellosis might be associated with disturbances
of IL-2/IL-2 receptor system [21].Stimulation
of human T-lymphocytes by LPS was found to be MHC-unrestricted, but strongly
dependent on costimulatory signals provided by CD80/CD28 interactions [42]. CD80 seems to be crucial for the
activation of T-cells by monocytes, since monocytes expressing CD86, but not
CD80 after LPS stimulation, were unable to stimulate human T-cells [42]. Moreover, endotoxin
administration to healthy subjects increases T-lymphocytes CD28 expression [32].Noteworthy, when PHA-cultured PBMCs were stimulated byE.
coli LPS, CB patients and especially CB1 subgroup (relapsing patients)
displayed significantly lower percentage of helper T-lymphocytes expressing
CD28 costimulation receptor in comparison to acutely ill patients, ranging
at the levels of controls. In our previous study [23], HKBA stimulation of
PHA-cultured PBMCs did not increase
the frequency of CD4+/CD28+ T-cells in chronic relapsing brucellosis (Table 3(b)).It
could be expected that a potent pathogen-associated molecule pattern (PAMP)
stimulation, either byE. coli LPS or HKBA, should lead to the upregulation
of CD28+ T-cells in CB1 patients. It seems that chronic relapsing brucellosis
patients are unresponsive/anergic to nonspecific or brucellar antigens as it
was shown previously [17, 19, 20, 43, 44]. On the other hand, the effect of E.
coli on PHA-cultured CD4+/CD28+ T-cells could be attributed to “LPS
tolerance phenomenon”, where prior
sublethal exposure to LPS results in a state of tolerance to further LPS
challenge [45]. LPS tolerance is a state of altered immunity characterized by
decreased production of macrophage-derived cytokines, such as TNFα, IL-1β,
and IL-6, as well as lymphocyte-derived IFNγ [46, 47]. As a result LPS tolerant,
macrophages have a markedly impaired ability to induce IFNγ secretion by autologous T-cells and NK-cells
[48]. It
could be assumed that in chronic relapsing brucellosis patients prior exposures
to Brucella LPS, as a result of relapses during the chronic stage, leads to “cross
tolerance” (low percentage of CD4+/CD28+ T-cells) to further in vitro E.
coli LPS challenge. Nevertheless, it is not known if chronic brucellosis
patients present high incidence of
gram negative enterobacteria infections.E.
coli addition in PHA cultures did not further enhance the increase in the
percentage of CD14+/CD80+ monocytes in both groups of brucellosis patients. The
percentage of CD14+/CD80+ cells with PHA plus LPS in PBMCs cultures were
similar to that obtained with PHA alone [23] (Figure 1). It could be speculated
that patients monocytes display maximal activation when exposed to PHA in
cultures and cannot be further activated by E. coli LPS addition. The
inflammatory microenvironment
of the infection may also contribute to this effect [49, 50]. Specifically, in
chronic brucellosis patients, high percentage of CD14+/CD80+ monocytes in PHA
and PHA plus E. coli LPS cultures might compensate for an ineffectual
CD4+ T-cell response characterizing these form of disease [17–21].Previously,
we stimulated PHA-cultured PBMCs from brucellosis patients with HKBA in order
to investigate any possible effect of brucellar antigen/s (mainly LPS) on the
expression of CD25 and CD80/CD28 costimulation molecules [23]. We used whole
cells of HKBA in PHA cultures in order to evaluate the immunomodulative potential
of the total brucellar antigenic structure. HKBA addition in comparison to E.
coli LPS stimulation led to a significant reduction of CD4+/CD25+
T-lymphocytes and CD14+/CD80+ monocytes PBMCs in a dose-related manner
supporting an immunomodulating effect of brucella on human PBMCs (Figure 1) [23].Actually,
in human macrophages, smooth LPS protects Brucella from
phagosome-lysosome fusion, bactericidal cationic peptides, and complement attack and,
moreover, is able to induce IL-10 (anti-Th1 cytokine) by PBMCs of healthy
individuals [8, 24, 51–55]. Interestingly, recent data suggest
that the smooth strains of living Brucella, in contrast to E. coli,
prevent the maturation of infected human dendritic cells and impair their
capacities to present antigen to naïve T-cells and to secrete IL-12 [16, 26]. Moreover, other results suggest that HKBA also inhibits major
histocompatibility complex class II expression and antigen processing in human
monocytes [56].In conclusion, T-lymphocytes of chronic brucellosis patients retain
low percentages of CD4+/CD25+ and CD4+/CD28+ T-lymphocytes after potent stimulation
of PHA-cultured PBMCs by E. coli LPS, although the frequency of
CD14+/CD80+ monocytes remains increased. Based on the findings revealed after
the comparison of the PBMCs PHA-cultures treated with specific (HKBA) or no
specific (E. coli LPS) antigen, we suggest that Brucella modulates both
functional arms (innate and adaptive) of human immune system, leading to T-cell
anergy and chronic infection.



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