# The Activation of Macrophage and Upregulation of CD40 Costimulatory Molecule in Lipopolysaccharide-Induced  Acute Lung Injury

Authors: Liang Dong; Shujuan Wang; Ming Chen; Hongjia Li; Wenxiang Bi
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

To study the activation of macrophage and upregulation of costimulatory molecule of CD40 in lipopolysaccharide- (LPS-) induced acute lung injury (ALI) model, and to investigate the pathogenecy of ALI, mice were randomly divided into two groups. ALI model was created by injecting 0.2 mg/kg LPS in phosphate saline (PBS) in trachea. The pathologic changes of mice lungs were observed by HE staining at 24 and 48 hours after LPS treatment, then the alveolar septum damage, abnormal contraction, alveolar space hyperemia, and neutrophils or other inflammatory cells infiltration in the LPS group, but not in the control group, were observed. The expression of CD40 mRNA and CD40 protein molecules were higher in LPS group as compared to the control group by Northern blot and flow cytometry, respectively. Expression of Toll-like receptor-4 (TLR4) in activated macrophage (AMΦ) was higher in LPS group as compared to the control group by RT-PCR. The activation of NF-κB binding to NF-κB consensus oligos increased in LPS group by EMSA in macrophage. The concentrations of TNF-α, MIP-2, and IL-1β cytokines from bronchoalveolar lavage fluid (BALF) were increased significantly in LPS group as compared to the control group by ELISA. The activation of AM and upregulation of costimulatory molecule CD40 induced all kinds of inflammatory cytokines releasing, then led to ALI. Therefore, both of them played vital role in the process of development of ALI.


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## Body

## 1. Introduction

It is well known that almost all of respiratory
diseases entail acute lung injury (ALI); it is the end result of common pathways initiated by a variety of local
or systemic insults leading to diffuse damage of the pulmonary parenchyma. Despite the
accumulation of abundant  information regarding the physiological and cellular
basis of lung injury and increasing sophisticated intensive care, an
improvement in prognosis has lagged behind. Therefore, the fatality rate of ALI
is higher and still remains as the major cause of mortality in intensive care
units (ICUs) [1]. It has become clear that there is not one mediator
responsible for ALI, but rather a complex interplay including some inflammatory
mediators. Lipopolysaccharide
(LPS) is a glycolipid that constitutes the major portion of outmost membrane of
gram-negative bacteria, and it
is capable of inducing severe lung injury in Gram-negative bacteria sepsis
and pneumonia, which are among the most common predisposing causes of ALI [2]. The interaction of the
lipid A moiety of LPS with macrophages, especially activated alveolar
macrophages (AMΦ) in the lung, appears
to be especially important because subsequent cellular activation results in
the release of inflammatory mediators and phenotypic changes [3, 4]. As
inflammatory mediators, AMΦ could release systemically active proinflammatory
cytokines and chemokines, including tumor-necrosis factor-α (TNF-α), Interleukin-1β
(IL-1β),  and macrophage inflammatory protein-2 (MIP-2) [5].CD40, a costimulatory molecule for
antigen presentation, is expressed by a wide variety of cells including
macrophages. Aberrant expression of CD40 is associated with autoimmune
inflammatory diseases. Interaction of Toll-like receptor-4 (TLR4) with the Gram-negative
bacteria endotoxin LPS results in the induction of an array of immune response genes [6, 7]. As numerous recent studies have demonstrated, the CD40 ligation
on APC is associated with the augmentation of inflammation as follow: secretion
of cytokines TNF-α, IL-1β, and MIP-2. A
great number of
studies indicate
that the CD40L on APC is
related to inflammatory amplification. The interaction of CD40-CDL in the lung
is a critical step to mediate the inflammation [8]. In fact, the mechanism of the activation of
macrophage and upregulation of CD40 costimulatory molecule in ALI is
undetermined. Because the natural
history of many of these diseases is unknown, animal model studies have been
undertaken to fill in the gaps and to provide important clues to their roles in
ALI.In this study, we describe that LPS
is a strong inducer of CD40 expression in macrophages in ALI
mice model, which occurs
at the transcriptional level and involves the activation of the transcription factors
nuclear factor-κB (NF-κB).
LPS-induced CD40 expression involves a lot of cytokines such as TNF-α,
IL-1β, and MIP-2.


## 2. Materials and Methods

Reagents were obtained as follows: E.coil
LPS was purchased from Sigma-Aldrich
(St. Louis, Mo, USA).
Enzyme-linked immunosorbent assay (ELISA) development kits for mouse TNF-α, IL-1β, and MIP-2
were obtained from Genzyme Techne Corporation (Minneapolis, Minn, USA). Anti-mouse TNF-α, MIP-2, and IL-1β signal clone antibiotics were from Sigma-Aldrich. Total cellular RNA
extraction and purification kits were purchased from Roche Corporation (Basel, Switzerland).
RT-PCR kits were obtained from Promage Corporation (Madison, Wis, USA). Rat anti-mouse PE-conjugated CD40 antibody (clone 3/23) and rat-PE IgG
isotype control were from PtarMigen (San
Diego, Calif, USA). Antibodies against CD40 and actin
were from Santa Cruz Biotechnology (Santa
Cruz, Calif, USA).


### 2.1. Induction of Animal Model

180 BALB/c wild-type (WT)
mice (6-to-8 week old) from Shandong University animal experiment center (Shandong, China) were divided two groups named as LPS group (B group) and control group
(A group), respectively. Mice were anesthetized with pentobarbital sodium
followed by injecting 0.2 mg/kg LPS in phosphate saline (PBS) intratrachealy for
group B and PBS alone for group A [9].


### 2.2. Collection and Measurement of Specimens

After
mice were executed, a total of 2.5 mL brochoalveolar lavage fluid (BALF) was
collected [10]. The trachea was cannulated, and the lungs were lavaged six times
with PBS (0.5 mL each time). Total cell numbers were counted with a standard
hemocytometer. After centrifugation, supernatants were stored −80°C for
cytokine measure by ELISA, and cell pellets were used to prepare cytospins.


### 2.3. Cell Culture

A smear of BAL cells was
prepared with cytocentrifugation using acytospin 2 at 1000 rpm for 5 minutes
and then stained with Giesma solution [11]. Cell differentiation was examined
by counting at least 200 cells using hemocytologic criteria to classify the
cells as neutrophils, eosinophils, lymphocytes, or macrophages. By morphologic
estimation with Gimesa staining, it was confirmed that polled cells from each
group consisted of >98% AMΦ for separate experiments. Cells were resuspended
in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/lstreptomycin,
0.25 μg/L amphotericcin B, and 10% fetal calf serum, which was used as a
complete medium. After allowing the cells to adhere to plates for 3 hours, nonadjacent
cells were removed with three washes. The remaining adhesive cells were used as
AMΦ. Macrophages were about 1×105/mL in
inverted microscope.


### 2.4. RNA Analysis by RT-PCR

Total cellular RNA was extracted
according to manufacture’s instruction. TLR4 primers were designed by Gene Toll (616 bp) forward5′TCCCACCACC
GATTCACA3′, reverse 5′CAACC2CTTTCATTTCACA3′. PCR reaction conditions were as follow: 94°C denature 2 minutes,
then 94°C denature 30 seconds, 58°C anneal 60 seconds, 68°C extend 2 minutes, after
40 cycles, 68°C extend 10 minutes. β-actin (200 bp) (5′TGGGTCAGAAGGACTCCTATGTG3′, reverse 5′CGTCCCAGTTGGTAACAATGC) was used as internal
control for quantification by densitometry.


### 2.5. ELISA Assay for TNF-α, MIP-2, and IL-1β

BALFs
were
collected as before, anti-mouse TNF-α, MIP-2, and IL-1β and
polyclonal horseradish peroxidase-conjugated IgG were sequentially added as
primary and secondary antibodies, respectively. The color was developed with
o-phenylenediamine substrates, and read with universal microplate reader.


### 2.6. EMSA for NF-κB

Nuclear
and cytoplasmic extracts were obtained
from cells from two groups. Protein level
was determined in all extracts using the Bio-Rad dye reagent assay (Hercules,
Calif, USA).
An equal amount (5μg) of nuclear protein from each sample and
NF-κB consensus oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′ (Promega)
was used for EMSA following the manufacture’s instructions as
previously described [12].


### 2.7. Northern Blot and Immunofluorescence Flow Cytometry
for CD40 mRNA and Protein

Total cellular RNA was
isolated at 24 and 48 hours after from two groups. Mouse CD40 and GAPAH probe
were prepared as described previously [13, 14]. 20 μg of total RNA was
hybridized with probes at 42°C  overnight. The hybridized mixture was treated with RNaseA/T1 (1 : 2000) and analyzed
by 5% denaturing polyacrylamide gel electrophoresis. Values for CD40 mRNA levels were normalized to
GAPDH mRNA level for each experiment condition.Cells were plated at2×105 cells/well
into 12 well plates after 48 hours, and then incubated with 100 μL of 2.4G2 hybridoma supernatant (which
contains rat-anti-mouse Fc r R
Ab) for blocking Fc r R.
Cells were then incubated with 10 μg/mL PE-conjugated anti-mouse CD40 Ab, and analyzed on the FACStar (Becton Dickinson, Mountain View, Calif, USA) [15]. Nagative controls were
incubated with IgG isotype-matched antibody. Fold induction of CD40 expression
was calculated by dividing the value of mean fluorescence intensity (MFI) of
regents treated samples by value of untreated samples.


### 2.8. Statistical Analysis

Data were presented as means ±SD. Continuous variables were tested by
analysis oft-test. The P-values are two-tailed and a P-value of less than .05 was considered statistically significant.


## 2.1. Induction of Animal Model

180 BALB/c wild-type (WT)
mice (6-to-8 week old) from Shandong University animal experiment center (Shandong, China) were divided two groups named as LPS group (B group) and control group
(A group), respectively. Mice were anesthetized with pentobarbital sodium
followed by injecting 0.2 mg/kg LPS in phosphate saline (PBS) intratrachealy for
group B and PBS alone for group A [9].


## 2.2. Collection and Measurement of Specimens

After
mice were executed, a total of 2.5 mL brochoalveolar lavage fluid (BALF) was
collected [10]. The trachea was cannulated, and the lungs were lavaged six times
with PBS (0.5 mL each time). Total cell numbers were counted with a standard
hemocytometer. After centrifugation, supernatants were stored −80°C for
cytokine measure by ELISA, and cell pellets were used to prepare cytospins.


## 2.3. Cell Culture

A smear of BAL cells was
prepared with cytocentrifugation using acytospin 2 at 1000 rpm for 5 minutes
and then stained with Giesma solution [11]. Cell differentiation was examined
by counting at least 200 cells using hemocytologic criteria to classify the
cells as neutrophils, eosinophils, lymphocytes, or macrophages. By morphologic
estimation with Gimesa staining, it was confirmed that polled cells from each
group consisted of >98% AMΦ for separate experiments. Cells were resuspended
in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/lstreptomycin,
0.25 μg/L amphotericcin B, and 10% fetal calf serum, which was used as a
complete medium. After allowing the cells to adhere to plates for 3 hours, nonadjacent
cells were removed with three washes. The remaining adhesive cells were used as
AMΦ. Macrophages were about 1×105/mL in
inverted microscope.


## 2.4. RNA Analysis by RT-PCR

Total cellular RNA was extracted
according to manufacture’s instruction. TLR4 primers were designed by Gene Toll (616 bp) forward5′TCCCACCACC
GATTCACA3′, reverse 5′CAACC2CTTTCATTTCACA3′. PCR reaction conditions were as follow: 94°C denature 2 minutes,
then 94°C denature 30 seconds, 58°C anneal 60 seconds, 68°C extend 2 minutes, after
40 cycles, 68°C extend 10 minutes. β-actin (200 bp) (5′TGGGTCAGAAGGACTCCTATGTG3′, reverse 5′CGTCCCAGTTGGTAACAATGC) was used as internal
control for quantification by densitometry.


## 2.5. ELISA Assay for TNF-α, MIP-2, and IL-1β

BALFs
were
collected as before, anti-mouse TNF-α, MIP-2, and IL-1β and
polyclonal horseradish peroxidase-conjugated IgG were sequentially added as
primary and secondary antibodies, respectively. The color was developed with
o-phenylenediamine substrates, and read with universal microplate reader.


## 2.6. EMSA for NF-κB

Nuclear
and cytoplasmic extracts were obtained
from cells from two groups. Protein level
was determined in all extracts using the Bio-Rad dye reagent assay (Hercules,
Calif, USA).
An equal amount (5μg) of nuclear protein from each sample and
NF-κB consensus oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′ (Promega)
was used for EMSA following the manufacture’s instructions as
previously described [12].


## 2.7. Northern Blot and Immunofluorescence Flow Cytometry
for CD40 mRNA and Protein

Total cellular RNA was
isolated at 24 and 48 hours after from two groups. Mouse CD40 and GAPAH probe
were prepared as described previously [13, 14]. 20 μg of total RNA was
hybridized with probes at 42°C  overnight. The hybridized mixture was treated with RNaseA/T1 (1 : 2000) and analyzed
by 5% denaturing polyacrylamide gel electrophoresis. Values for CD40 mRNA levels were normalized to
GAPDH mRNA level for each experiment condition.Cells were plated at2×105 cells/well
into 12 well plates after 48 hours, and then incubated with 100 μL of 2.4G2 hybridoma supernatant (which
contains rat-anti-mouse Fc r R
Ab) for blocking Fc r R.
Cells were then incubated with 10 μg/mL PE-conjugated anti-mouse CD40 Ab, and analyzed on the FACStar (Becton Dickinson, Mountain View, Calif, USA) [15]. Nagative controls were
incubated with IgG isotype-matched antibody. Fold induction of CD40 expression
was calculated by dividing the value of mean fluorescence intensity (MFI) of
regents treated samples by value of untreated samples.


## 2.8. Statistical Analysis

Data were presented as means ±SD. Continuous variables were tested by
analysis oft-test. The P-values are two-tailed and a P-value of less than .05 was considered statistically significant.


## 3. Results

### 3.1. Pathologic Changes of Lung Tissue

Compared
with mice in control group, the lung tissue from LPS-treated mice demonstrated interalveolar
septum collapse,
abnormal constraction, alveolar space and interstitial congestion, and neutrophilic
granulocyte infiltration (Figure1).Histopathological
examination. Three pictures of pathological sections showing (a) pathological appearance from control group, (b) pathological appearance from LPS group at
24 hours, and (c) pathological appearance
from LPS group at 48 hours(HE*300).
(a)(b)(c)


### 3.2. TLR4 Expresses in Macrophages

The gene expression level of TLR4 in AMΦ detected by RT-PCR is higher in LPS group as compared to the controls
(Figure 2).Figure 2
mRNA
expression of TLR4
in AMΦ by RT-PCR. A representative agarose gel electrophoresis showing
PCR amplification of TLR4 and β-actin from cultured AMΦ with control group and LPS group (at
24, 48 hours).


### 3.3. LPS Induces NF-κB Binding to the NF-κB Consensus Oligos

To investigate the role of LPS in regulation
of transcription factor activity, NF-κB activity of macrophage in control and
LPS groups were compared. LPS stimulation led to increased NF-κB binding to
NF-κB consensus oligos as demonstrated by EMSA (Figure 3).Figure 3
Activation of NF-κB by EMSA. NF-κB
activity of macrophage in control and LPS groups (at 24, 48 hours).


### 3.4. The Expression Levels of CD40 mRNA and Protein

The
expression level of CD40 mRNA was determined by Northern blot as shown in Figure4(a). LPS-induced CD40 mRNA expression increased in both 24 and 48 hours LPS group
as compared to the control group. The
expression level of CD40 protein determined by immunofluorescence
flow cytometry increased after 48 hours in LPS group as compared to the control
group (Figure 4(b)).mRNA and protein expression of CD40 in AMΦ by RT-PCR and flow cytometry, respectively, (a) a representative agarose gel electrophoresis
showing PCR amplification of CD40
andβ-actin from cultured AMΦ with control group and LPS group (at
24, 48 hours),  (b) protein expression of CD40 in AMΦ with control group and LPS group.
(a)(b)


### 3.5. The Expression Levels of TNF-α, MIP-2, and IL-1β in LPS-Induced Model

The inflammatory cytokine levels in BALF determined by ELISA
demonstrated an increased expression of
TNF-α, MIP-2, and IL-1β
in LPS group as compared to the control
group (Table 1, Figure 5).Table 1
Concentration of TNF-α, MIP-2, and IL-1β cytokines from BALF (x¯±s).
GroupnTNF-α (pg/mL)IL-1β (pg/mL)MIP-2 (pg/mL)Control group1061.02±10.710.7±2.784.5±15.8LPS group10115.0±29.6*302.1±17.3*524.9±99.1*(P<.05*, compared with the control group).Concentration of TNF-α, MIP-2, and IL-1β cytokines
from BALF: (a) concentration of TNF-α, (b) concentration of MIP-2, (c) concentration of IL-1β.
(a)(b)(c)


## 3.1. Pathologic Changes of Lung Tissue

Compared
with mice in control group, the lung tissue from LPS-treated mice demonstrated interalveolar
septum collapse,
abnormal constraction, alveolar space and interstitial congestion, and neutrophilic
granulocyte infiltration (Figure1).Histopathological
examination. Three pictures of pathological sections showing (a) pathological appearance from control group, (b) pathological appearance from LPS group at
24 hours, and (c) pathological appearance
from LPS group at 48 hours(HE*300).
(a)(b)(c)


## 3.2. TLR4 Expresses in Macrophages

The gene expression level of TLR4 in AMΦ detected by RT-PCR is higher in LPS group as compared to the controls
(Figure 2).Figure 2
mRNA
expression of TLR4
in AMΦ by RT-PCR. A representative agarose gel electrophoresis showing
PCR amplification of TLR4 and β-actin from cultured AMΦ with control group and LPS group (at
24, 48 hours).


## 3.3. LPS Induces NF-κB Binding to the NF-κB Consensus Oligos

To investigate the role of LPS in regulation
of transcription factor activity, NF-κB activity of macrophage in control and
LPS groups were compared. LPS stimulation led to increased NF-κB binding to
NF-κB consensus oligos as demonstrated by EMSA (Figure 3).Figure 3
Activation of NF-κB by EMSA. NF-κB
activity of macrophage in control and LPS groups (at 24, 48 hours).


## 3.4. The Expression Levels of CD40 mRNA and Protein

The
expression level of CD40 mRNA was determined by Northern blot as shown in Figure4(a). LPS-induced CD40 mRNA expression increased in both 24 and 48 hours LPS group
as compared to the control group. The
expression level of CD40 protein determined by immunofluorescence
flow cytometry increased after 48 hours in LPS group as compared to the control
group (Figure 4(b)).mRNA and protein expression of CD40 in AMΦ by RT-PCR and flow cytometry, respectively, (a) a representative agarose gel electrophoresis
showing PCR amplification of CD40
andβ-actin from cultured AMΦ with control group and LPS group (at
24, 48 hours),  (b) protein expression of CD40 in AMΦ with control group and LPS group.
(a)(b)


## 3.5. The Expression Levels of TNF-α, MIP-2, and IL-1β in LPS-Induced Model

The inflammatory cytokine levels in BALF determined by ELISA
demonstrated an increased expression of
TNF-α, MIP-2, and IL-1β
in LPS group as compared to the control
group (Table 1, Figure 5).Table 1
Concentration of TNF-α, MIP-2, and IL-1β cytokines from BALF (x¯±s).
GroupnTNF-α (pg/mL)IL-1β (pg/mL)MIP-2 (pg/mL)Control group1061.02±10.710.7±2.784.5±15.8LPS group10115.0±29.6*302.1±17.3*524.9±99.1*(P<.05*, compared with the control group).Concentration of TNF-α, MIP-2, and IL-1β cytokines
from BALF: (a) concentration of TNF-α, (b) concentration of MIP-2, (c) concentration of IL-1β.
(a)(b)(c)


## 4. Discussion

In this study, we demonstrated that the levels of inflammatory
cytokines TNF-α, MIP-2, and IL-1β increased
significantly in BALF in LPS-induced ALI mice
model. Such cytokines have been studied
more clearly than others in ALI, which have played critical role in
inflammation development process. MIP-2, a functional analog of human IL-8, is
an important mediator in the recruitment of neutrophils, and TNF-α acts locally to stimulate chemotaxis and activate neutrophils.
These cytokines might result in the injury of the lung and eventually the
development of ALI to some extent [16].LPS
can induce increased gene expressions in AMΦ, such as TLR4, CD40, TNF-α, MIP-2, and IL-1β, and activate some
signal transduction pathways such as TLR4-mediated NF-κB activation, and CD40-CD40L
interaction. These changes will result subsequently in  releasing of large amount of
inflammatory cytokines [17]. AMΦ was the first cell type in the primary
immunity to kill bacterium after infection [18]. They are also the target cells
to LPS. Organisms can release a large number of inflammatory and anti-inflammatory
cytokines in endotoxemia [19]. TLR4 is a kind of transmembrane receptor on LPS target
cell in immunity system [20]. It mediates the signal of LPS from outside to inside cell. From our experiments, we observed that TLR4 mRNA and TNF-α enhanced significantly in
AMΦ of LPS-induced ALI. TLR4 is then regarded as
the specific recognition receptor in the process of development ALI. The lung tissue
was probably injured through increased secretion of TLR4 and strengthening its
signal pathway. Therefore, TLR4 plays a vital role in a series of signals activation.
LPS-TLR4 compound is able to activate many kinds of signals activation [21]: TLR4-induced
NF-κB
activation promotes
the CD40-CD40L pathway as well as the CD40 self-activation, thus it causes the massive
inflammation factors release and expanded inflammation response.Previous reports demonstrated that the
LPS-TLR4 interaction activated NF-κB directly. Subsequently, they all bind to the promoter of CD40, finally
activated the CD40-CD40L interaction pathway and the CD40 self-activation, led
to the release of inflammation factors and the anti-inflammation factors, which
activated the acquired immunity. Therefore, the CD40 activation played a vital role
in the process of development of ALI. In our study, we demonstrated that CD40 mRNA
and the protein level increased in the macrophage in LPS group.Large body of literatures demonstrated the expression of CD40L in the T
lymphocytes and mast cells. Activation of CD40-CD40L pathway may promote the
massive inflammation factors release, and the CD40-CD40L
activation also indicates that T-cell dependence immunity already started [22, 23],
which subsequently upregulated the other costimulatory molecules and the cell
adhesion molecules to promote the massive preinflammation cell factor
production and release, as well as inflammation cell differentiation. Our data
demonstrated high concentrations of TNF-α, MIP-2, and IL-1β cell factors in
BALF from LIP group, indicating that the CD40-CD40L pathway can enhance the inflammation
response and gather massive inflammation cells, finally induces and aggravates
the inflammation extent of lung injury.


## 5. Conclusions

Our data demonstrated that LPS-induced CD40 gene transcription, activated
NF-κB transcription
factors, increased their affinity and finally bound to the promoter of CD40, which
led to the high expression of CD40 in the acute lung injury. Studying the mechanism of
LPS-induced, TLR4-mediated in vitro CD40 transcription allowed us to provide
the experimental evidences of the important role of CD40 in the development of
LPS-induced ALI. We thus predict that blocking the expression of TLR4 or the inhibition of expression of CD40 through interruption
of its transcription regulation complexes will be attractive targets to the
treatment of LPS-induced ALI.



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*Source: 852571-2008-04-20.xml*