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C57Bl/6 and BALB/c Mice Have Similar Neutrophil Response to Acute Streptococcus pneumoniae Sinus Infections
Usama Gabr, MD;
Yu-Sung Won, MD, PhD;
Sue Boonlayangoor, MS;
Kenneth Thompson, PhD;
Fuad M. Baroody, MD;
Robert M. Naclerio, MD
Arch Otolaryngol Head Neck Surg. 2001;127:985-990.
ABSTRACT
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Background Previous investigations have shown that mice with a tendency toward
a TH1 or TH2 lymphocyte response manifest different
reactions to inoculation with the parasite Leishmania major. BALB/c mice (with a tendency for a TH2 response) showed
evidence of systemic infection, whereas C57Bl/6 mice (with a tendency for
a TH1 response) showed only a local reaction.
Objective To investigate whether BALB/c and C57Bl/6 mice respond differently to
acute bacterial infection of the sinuses.
Methods We inoculated the nasal cavities of C57Bl/6 and BALB/c mice with Streptococcus pneumoniae (type ATCC59), or with broth as
a control. The mice were humanely killed 2, 5, 10, and 14 days after inoculation.
Their heads were fixed, decalcified, and embedded in paraffin blocks. Sections
were stained with hematoxylin and eosin, and the degree of inflammation was
quantified by the number of neutrophils per square millimeter of the sinus
mucosa and the percentage of the sinus cavity occupied by neutrophil clusters.
Results Both groups of mice showed evidence of inflammation that was significantly
greater than controls (P = .01), with no difference
between groups. There was a correlation between the number of neutrophils
per square millimeter in the sinus mucosa and the percentage of neutrophil
clusters (C57Bl/6 mice, r = 0.37, P<.001; BALB/c mice, r = 0.20, P<.001). In the infected mice, the number of infiltrating neutrophils
was significantly greater (P<.001) in anatomically
lower (dependent) areas of the sinuses compared with the upper areas.
Conclusion Unlike leishmaniasis, acute bacterial sinusitis is not affected by the
tendency of the host to favor either a TH1 or TH2 response.
INTRODUCTION
ACUTE BACTERIAL infection of the sinuses is a common disorder that is
often preceded by a viral upper respiratory tract infection. During acute
infection, Streptococcus pneumoniae is the most common
bacterium obtained from maxillary sinus punctures.1-2 Streptococcus pneumoniae has been studied extensively,
and its complete genome sequence has been identified.3
Several virulent factors affect the host's response to this organism, including
(a) protein adhesion molecules (attach to the N-acetylgalactose complex of the respiratory mucosa),4 (b) IgA protease proteins
(inactivate respiratory mucosal IgA),5 (c) antiphagocytic polysaccharide capsules (interfere with
the opsonizing activity of the alternative complement pathway),3
(d) cell wall lipotechoic acid (a potent proinflammatory
complex capable of eliciting the production of interleukin 1 (IL-1) and tumor
necrosis factor  in addition to the activation of the alternative complement
pathway),3 and (e)
pneumolysin toxin (has complement-activating and direct cytolytic activities).3, 6-7 However, far less attention
has been focused on the host's immune response to infection.
Basic immunologic research has demonstrated functional differences between
the 2 T-helper lymphocyte subsets. TH1 cells produce IL-2 and interferon 
(IFN-), which are important in the cell-mediated immune response directed
toward the production of IgG-subclass antibodies (IgG1 and IgG2). On the other
hand, TH2 cells respond by producing IL-4, IL-5, IL-10, and IL-13,
which help to create a microenvironment favoring eosinophil-mediated inflammatory
responses and the production of IgE and IgA antibodies.8
Moreover, there is reciprocal inhibition (down-regulation) between the TH1 and TH2 lymphocytes secondary to their cytokine production
(ie, TH1 cytokines inhibit the development of TH2 cells,
and vice versa). Disturbances in the balance between TH1 and TH2 and the cytokines that they produce may exist and contribute to the
pathogenesis of inflammatory diseases.
In mice, several studies9-12
have shown that the genetic tendency of the animal to favor a TH1
or TH2 response plays a major role in determining the course and
severity of parasitic infection with Leishmania major.
C57Bl/6 mice, with a TH1 tendency, show resistance to systemic
infection and consistently produce high levels of IFN-13
and a localized inflammatory reaction. In contrast, BALB/c mice, with a TH2 tendency, develop a disseminated and lethal infection. In a model
of visceral leishmaniasis, a similar study14
using intravenous injection with Leishmania infantum
showed no differences in the response between the 2 strains of mice.
In human rhinosinusitis, attempts have been made to characterize the
cytokine profile of different types of sinus mucosal pathologic conditions15-21
and the role of allergy in sinus disease.22
Hamilos et al,15 in 1995, showed evidence for
different cytokine expression in allergic vs nonallergic patients with chronic
sinusitis and found a preponderance of TH2-type cytokines in both
groups. Other authors16 also described up-regulation
of mRNA expression of proinflammatory TH2 cytokines in chronic
hyperplastic sinusitis with nasal polyposis. Moreover, recent studies17-20 suggest
different cytokine profiles for different diseases, in which acute sinusitis
is associated with IL-8 levels, nasal polyposis with IL-5, and chronic sinusitis
with IL-3. These investigations emphasize the potential role of different
T-lymphocyte subsets and their contribution to the pathophysiology of sinus
disorders.
The aim of our study was to determine whether the genetic tendency of
the host to favor either a TH1 or TH2 response would
affect the inflammatory reaction to S pneumoniae
infection of the paranasal sinuses in mice.
MATERIALS AND METHODS
MICE
Black C57Bl/6 and white BALB/c mice aged 6 to 8 weeks were obtained
pathogen-free from The Jackson Laboratory, Bar Harbor, Me, and housed in micro-isolation
cages at the Carlson Biocontainment Facility at The University of Chicago,
Chicago, Ill. All manipulations of the animals before they were humanely killed
were conducted in a class II biosafety hood, following strict biosafety control
measures as outlined by the university's Animal Resource Center. The Animal
Safety Committee of The University of Chicago approved the study.
DESIGN
On day 0 (the start of the experiment), the mice were given an intranasal
inoculation of S pneumoniae in broth, and they were
then killed on days 2, 5, 10, and 14. On each day of carnage, 1 group of C57Bl/6
mice and 1 group of BALB/c mice were included. Thirty-seven C57Bl/6 mice and
35 BALB/c mice completed the study. In addition, a control group of 4 BALB/c
mice were challenged with broth only and were killed on day 5 after inoculation.
The number of mice in the control group was small and consisted of only BALB/c
mice because prior investigation showed no response to the broth in noninfected
C57Bl/6 mice.23
INOCULA
All inocula were freshly prepared in the clinical microbiology laboratory
of The University of Chicago Medical Center. Streptococcus
pneumoniae (type ATCC59) was suspended in trypticase soy broth at a
3.0 McFarland standard concentration equivalent to about 1.2 x 109 colony-forming units per milliliter. Mice in the control
group were challenged with trypticase soy broth only.24
METHODS
The animals were sedated with ketamine hydrochloride (80 mg/kg of body
weight) and xylazine hydrochloride (8 mg/kg of body weight) by intraperitoneal
injection. Their head was tilted backward, and 5 droplets of the inoculum
were placed at the anterior nares of each nostril. Because mice are obligatory
nasal breathers, the fluid was drawn into the nasal passages during inhalation,
filling the nasal cavity and the sinus areas. To avoid choking the animal,
we placed inoculum droplets very slowly, with close monitoring of the breathing
rate. Animals were placed on their side until they recovered from anesthesia
in about 15 to 20 minutes. They were then housed in a pathogen-free facility
until they were killed.
TERMINATION PROCEDURE
On the designated day of carnage, the animals were euthanized with an
intraperitoneal injection of sodium pentobarbital (120 mg/kg of body weight).
While respiratory failure was in progress, the abdominal cavity was incised
transversely, exposing the abdominal surface of the diaphragm, which was quickly
opened for access to the chest cavity and the still-beating heart. A 21-gauge
blunt butterfly catheter was introduced into the apex of the left ventricle,
with care taken not to injure the interventricular septum; then, an incision
was made in the right atrium, and 80 to 100 mL of lactated Ringer solution
was pushed through the catheter for removal of blood, as determined by blanching
and blood no longer draining from the atrial incision. Next, the mouse was
perfused with about 50 mL of fixative solution (4% paraformaldehyde with 0.5%
glutaraldehyde in 0.1M phosphate buffer).
HISTOLOGIC PREPARATION
Under low magnification, the skin, muscles, and eyeballs were removed
from the decapitated head; the remaining tissue was immersed in the fixative
solution for 24 hours. Then, the mandible, tongue, and any remaining muscles
were removed. The remaining tissue was then immersed in a decalcifier solution
(Surgipath II, Rockford, Ill) for 24 hours. After decalcification, the heads
were softened enough to trim the anterior portion of the nose and the posterior
portion of the skull with the brain. The remaining block was embedded in paraffin,
and 5-µm-thick sections were cut. The sections were mounted on glass
slides and stained with hematoxylin-eosin.
CYTOLOGIC ANALYSIS
Three anatomically similar sections were selected from each mouse head,
at the middle portion of the sinus cavity. The sections included the posterior
end of the maxillary sinuses and the ethmoid sinuses; an arbitrary transverse
line at the level of the middle turbinate was used to divide the section into
upper and lower portions (Figure 1).
Investigators were blinded to the source groups of mice before analysis of
sections. A computer-assisted microscope (x40 lens) powered with image
analysis software (Neurolucida version 2.1; MicroBrightField Inc, Colchester,
Vt) was used for the analysis. The 2 variables assessed were the percentage
of the cross-sectional sinus cavity area occupied by neutrophil clusters (Figure 2) and the number of neutrophils per
square millimeter infiltrating the sinus mucosa (Figure 3). The infiltrating neutrophils were counted in areas adjacent
to the neutrophil clusters, or in 4 equivalent mucosal areas when no neutrophil
clusters were present.
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Figure 1. Microscopic tracing of the anatomic
structures of the mouse nasal and sinus cavities showing the maxillary sinus
(M), ethmoid sinus (E), nasal septum (S), and turbinates (T). An arbitrary
transverse division line is used (original magnification x5).
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Figure 2. Light micrograph of a coronal
section of the mouse ethmoid sinuses showing 2 neutrophil clusters (arrows)
filling a large portion of the cavity (hematoxylin-eosin, original magnification
x20).
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Figure 3. Light micrographs of the sinus
mucosa. A, Sinus mucosa from a control mouse. B, Inflamed mucosa in an infected
mouse, with dilated vessels (arrow), many neutrophil infiltrates, and mucosal
stripping (hematoxylin-eosin, original magnification x40).
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STATISTICAL ANALYSIS
Parametric statistical methods were used. Nonpaired t tests were applied to the data for comparison of the difference between
the 2 groups of mice killed on the same day, and for comparison of differences
between each group and control on day 5. We performed paired t-test analysis on the data between upper and lower areas of the mucosa
to determine whether the anatomic distribution of the infiltrates varied within
the same section. Numbers are expressed as mean ± SEM. P<.05 was considered to indicate statistical significance. The Pearson
product moment correlation test was applied for evaluation of the relationship
between the 2 objective variables used in the study.
RESULTS
Neither group of infected mice showed clinical symptoms of infection;
however, both groups showed neutrophil infiltrates and clusters significantly
greater in number than those in controls. This evidence of inflammation was
significant as early as day 2 of the experiment (P
= .01). Thereafter, the data showed an upward trend in the number of neutrophil
infiltrates that reached its peak on day 10 and then declined. The inflammation
was not totally resolved on day 14 (last day of the experiment). There was
no statistically significant difference between groups of mice killed on the
same day in either number of neutrophils or percentage of sinus cavity area
occupied by clusters (Figure 4 and Figure 5).
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Figure 4. Comparison of polymorphonuclear
neutrophils (PMNs) per square millimeter of infected C57Bl/6 and BALB/c mice.
There were significantly more PMNs per square millimeter of mucosa in the
infected groups on days 2, 5, 10, and 14 compared with the control group (P = .01). There were no significant differences between the 2 strains
on any day of the experiment. Data are presented as mean ± SEM.
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Figure 5. Comparison of polymorphonuclear
neutrophil (PMN) clusters of infected C57Bl/6 and BALB/c mice. None of the
mice in the control group showed clusters. The infected mice had significantly
more clusters in the sinus cavities than did the control group (P
= .01). There were no significant differences between the 2 mouse strains
at any time point. Data are presented as mean ± SEM.
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There was a significant correlation between the number of polymorphonuclear
neutrophils (PMNs) per square millimeter infiltrating the mucosa and the sinus
cavity area occupied by neutrophil clusters in both strains (C57Bl/6 mice, r = 0.37, P<.001; BALB/c mice, r = 0.20, P<.01) (Figure 6).
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Figure 6. The number of neutrophil infiltrates
in the mucosa correlates with the percentage of the sinus cavity occupied
by neutrophil clusters in both mouse strains. The top and bottom lines represent
95% confidence intervals and the middle line represents the regression line.
A, BALB/c mice, r = 0.20, P=<.01. B, C57Bl/6
mice, r = 0.37, P<.001.
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In both strains of mice and at all time points evaluated, the number
of PMNs infiltrating the mucosa showed significantly higher numbers in the
anatomically lower regions of the sinuses (dependent areas) compared with
the upper areas on the same section (P<.001) (Figure 7).
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Figure 7. The number of polymorphonuclear
neutrophils (PMNs) per square millimeter infiltrating the mucosa was significantly
higher in the lower (dependent) areas of the sinuses than in the upper areas
(P<.001) of the same sections. Data are depicted as mean (circles)
with SD (bars) and SEM (boxes).
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COMMENT
In the United States, sinusitis affects about 15% of the population.1 Sinusitis diminishes patients' quality of life, and
its treatment expends a large amount of health care resources. In 1992, the
estimated direct cost for the treatment of sinusitis reached $2.4 billion;
by 1996, the estimate was $5.8 billion.1 Despite
these facts, the pathophysiology of this disease is poorly understood. Consequently,
we developed a murine model of acute sinusitis in an attempt to better understand
the underlying mechanisms of the host involved in the pathophysiology and
immunology of this inflammatory disease.23
The immune system is complex and has many components that interact to
eliminate foreign invaders. This defense is imposed by nonspecific mechanisms
(innate immunity) and antigen-specific mechanisms (acquired immunity). Innate
immunity is present at birth and consists of intact skin and mucous membrane;
secretions and their components, macrophages, neutrophils, eosinophils, and
natural killer cells; the lactoferrin barrier; and isoenzymes.
The response of a naive host to bacteria begins with the innate immune
system, in which PMNs and macrophages are recruited. The macrophages act as
antigen-presenting cells, and they process, recognize, and present the foreign
antigens to other immune cells, particularly T-helper cells that continue
the acute inflammatory cascade. Because PMNs play such an important role in
the response to acute bacterial infection, we chose to quantify the number
of PMNs infiltrating the mucosa and the percentage of the sinus cavity occupied
by PMN clusters to assess the acute inflammatory response objectively. Although
involved and time-consuming, this method provides reliable and reproducible
data.
Neutrophil recruitment depends on chemotactic factors related to the
host and the bacteria. The results of this study suggest that the time course
and the degree of inflammation were not dependent on the genetically determined
differences between the 2 mouse strains. The difference between the 2 mouse
strains in response to cutaneous leishmaniasis9-14
led us to investigate whether a similar difference would be seen in acute
sinusitis. Our data suggest that this is not the case. Leishmania infection
is an IgE-mediated phenomenon, and TH2 lymphocytes are critical
to this process by virtue of the cytokines that they secrete, primarily IL-4
and IL-13, which are crucial for IgE synthesis. However, we do not know the
importance of T lymphocytes and their cytokines in acute bacterial infection
of the sinuses. The lack of difference in acute bacterial infection of the
sinuses between the 2 mouse strains with different TH lymphocyte
preponderance may suggest that TH cells are not central or critical
to this process. This may be explained in part by the strong ability of S pneumoniae to attract neutrophils independently of the
host's immune system. This ability is attributed to the T-independent antigens,
which include pneumolysin, the polysaccharide capsule, and lipoteichoic acid.
These antigens may act as the initiating factors for neutrophil recruitment
during acute infection. Moreover, they can activate the alternative complement
pathway and produce a primary IgM immune response without producing memory
cells or an anamnestic secondary IgG immune response.3-7
Accordingly, these groups of antigens can directly initiate acute inflammation,
independently of the host's genetic tendency toward either a TH1
or TH2 immune profile.5
Another possible explanation for our finding may be related to the immune
mediators released locally at the infection site, which act also as chemoattractants
to neutrophils; however, their release is not dependent on T-helper cells.
These mediators include complement component C5a, high-molecular-weight neutrophil
chemotactic factor, platelet-activating factor, IL-1 and IL-8, and leukotrienes,
such as leukotriene B4.
Our data suggest that the direct interplay between the bacterial antigens
and the local immune mediators may have an effect on the degree of acute bacterial
inflammation of the paranasal sinuses, more than that of the genetic tendency
toward either a TH1 or TH2 immune response. Furthermore,
we recognize that major differences may exist between acute and chronic inflammation
in regard to previous exposure to bacteria, antigen-specific antibody production,
and different cytokine profiles.17-21
Therefore, the host's tendency to favor a TH1 or TH2
response in chronic sinusitis may be an important determinant of the inflammatory
response, but remains to be investigated.
CONCLUSIONS
Unlike cutaneous leishmaniasis, acute bacterial sinusitis is not affected
by the genetic tendency of the mouse to favor either a TH1 or TH2 response. We speculate that the key factors affecting the course
of this disease are probably dependent on the direct antigenic nature of the
causative organism and its ability to stimulate the host's immune system within
the local environment.
AUTHOR INFORMATION
Accepted for publication February 2, 2001.
This work was supported by grants DC 02714 and AI 45583 from the National
Institutes of Health, Bethesda, Md.
Presented at the 56th Annual Meeting of the American Academy of Allergy,
Asthma, and Immunology, San Diego, Calif, March 6, 2000.
Corresponding author: Robert M. Naclerio, MD, Section of Otolaryngology–Head
and Neck Surgery, The University of Chicago, 5841 S Maryland Ave, MC 1035,
Chicago, IL 60637 (e-mail: rnacleri{at}surgery.bsd.uchicago.edu).
From the Sections of Otolaryngology–Head and Neck Surgery (Drs
Gabr, Won, Baroody, and Naclerio), and Clinical Microbiology (Ms Boonlayangoor
and Dr Thompson), Pritzker School of Medicine, The University of Chicago,
Chicago, Ill.
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