By
From the * Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115; and Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030
To determine the role of CD11/CD18 complexes in neutrophil emigration, inflammation was
induced in the skin, lungs, or peritoneum of mutant mice deficient in CD18 (CD18/
mutants). Peripheral blood of CD18
/
mutants contained 11-fold more neutrophils than did
blood of wild-type (WT) mice. During irritant dermatitis induced by topical application of
croton oil, the number of emigrated neutrophils in histological sections of dermis was 98% less
in CD18
/
mutants than in WT mice. During Streptococcus pneumoniae pneumonia, neutrophil
emigration in CD18
/
mutants was not reduced. These data are consistent with expectations based on studies using blocking antibodies to inhibit CD11/CD18 complexes, and on observations of humans lacking CD11/CD18 complexes. The number of emigrated neutrophils in
lung sections during Escherichia coli pneumonia, or in peritoneal lavage fluid after 4 h of S. pneumoniae peritonitis, was not reduced in CD18
/
mutants, but rather was greater than the WT
values (240 ± 30 and 220 ± 30% WT, respectively). Also, there was no inhibition of neutrophil emigration during sterile peritonitis induced by intraperitoneal injection of thioglycollate
(90 ± 20% WT). These data contrast with expectations. Whereas CD11/CD18 complexes are essential to the dermal emigration of neutrophils during acute dermatitis, CD18
/
mutant
mice demonstrate surprising alternative pathways for neutrophil emigration during pneumonia
or peritonitis.
Acute emigration of neutrophils requires CD11/CD18
complexes under most circumstances (for review see reference 1). Antibodies against CD11/CD18 inhibit neutrophil emigration during acute inflammation in animals (2, 3).
Leukocytes from human patients with leukocyte adhesion
deficiency type 1 (LAD-1),1 a disease arising from mutations in the gene for CD18, lack CD11/CD18 complexes.
In LAD-1 patients, neutrophil emigration is not observed using Rebuck skin windows or skin chambers (4), and
infected peritoneal, laryngeal, esophageal, periodontal, gingival, pharyngeal-glottic, dermal, or umbilical tissues of
LAD-1 patients are devoid of emigrated neutrophils (4, 5, 8).
In the lung, neutrophil emigration occurs via CD11/
CD18-dependent, but also via CD11/CD18-independent
pathways. Antibodies against CD11 or CD18 inhibit neutrophil emigration in response to Escherichia coli, E. coli LPS,
Pseudomonas aeruginosa, phorbol ester, IgG immune complexes, or IL-1 (3, 9), but these antibodies do not inhibit pulmonary emigration in response to Streptococcus pneumoniae, hydrochloric acid, or C5a complement fragments (3, 10). In contrast to the other tissues examined, the
lungs from an autopsied LAD-1 patient contained emigrated neutrophils (8).
Mice deficient in CD18 (CD18 Animals.
The CD18 gene was targeted for disruption in embryonic murine stem cells using a previously published targeting
construct (13), blastocysts containing mutant stem cells were
transferred to murine foster mothers, and a homozygous mouse
line was established by selective breeding, as previously described
(14). Mouse genotypes were confirmed by Southern blotting.
Wild-type mice were from the same genetic background (mixed
129/Sv and C57BL/6). Mice were studied at 8-18 wk of age. All
experiments received institutional approval.
CD11/CD18 Expression.
Expression of CD11/CD18 on circulating neutrophils was examined by flow cytometry. Mice were
killed by a lethal overdose of halothane. Blood was collected from
the inferior vena cava, erythrocytes were hypotonically lysed, and
leukocytes were stained with saturating concentrations of the following rat monoclonal antibodies from PharMingen (San Diego,
CA): M17/4 (anti-murine CD11a), M1/70 (anti-murine CD11b), or H129.19 (anti-murine CD4, used as a control antibody, nonbinding for neutrophils). Antibodies against CD11a and
CD4 were directly conjugated to FITC. Antibodies against
CD11b were biotinylated, and CD11b-labeled cells were secondarily labeled by streptavidin conjugated to FITC (PharMingen).
Cells were fixed with 1% paraformaldehyde, and then green fluorescence of 5,000 cells in the neutrophil population (identified
and gated using scatter profiles) was measured using an Ortho
Cytofluorograf 50HTM flow cytometer equipped with a Cicero
interface system (Cytomation, Fort Collins, CO).
/
mutants) have now
been derived (Scharffetter-Kochanek, K., submitted for publication). Neutrophils from these mice do not express
CD11/CD18 adhesion complexes. CD18
/
mutants display
a phenotype resembling that seen in humans with LAD-1,
including neutrophilia, peripheral lymphadenopathy, splenomegaly, and skin lesions. In this paper, CD18
/
mutant
mice were used to determine the roles of CD11/CD18
complexes in neutrophil emigration during inflammation
in the skin, lungs, or peritoneum.
Dermatitis. Irritant dermatitis was induced by topical application of croton oil. Mice were anesthetized by methoxyflurane inhalation, and each side of one ear was treated with 10 µl of 2% croton oil (Sigma Chemical Co., St. Louis, MO) in 4:1 acetone/ olive oil. After 6 h, mice were killed by an overdose of halothane inhalation. Ear widths were measured using spring-loaded calipers. Peripheral blood was collected from the inferior vena cava. Blood leukocytes were counted with a hemacytometer after erythrocyte lysis, and leukocyte differentials were counted in blood smears stained with LeukoStat (Fisher Scientific Co., Pittsburgh, PA). Each ear was removed, fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for examination by light microscopy.
Morphometric analysis was used to quantify neutrophil emigration using a drawing tube to reflect a grid onto microscopically viewed histologic sections. For each ear, the volume densities of emigrated neutrophils within four 110-µm-wide cross-sections, separated by 1.5-mm intervals, were assessed by point counting (18). A total of 547-1,677 points/ear were counted. Each point was assessed as falling on (a) epidermis, dermis, or cartilage, and (b) an emigrated neutrophil or not an emigrated neutrophil. The volume fraction of emigrated neutrophils in each ear was standardized to the cartilage volume of that ear, a value not expected to change during acute (6-h) dermatitis, by dividing the neutrophil volume fraction by the cartilage volume fraction. Cutaneous edema was quantified as the percentage of increase in ear width in the right (croton oil-treated) compared to the left (untreated) ear for each mouse. The width of each ear was measured five times with spring-loaded calipers. Edema (expressed as percentage swelling) was calculated as 100 times the difference in ear widths divided by the width of the untreated ear.Pneumonia. Pneumonias were induced by intratracheal instillation of bacteria, as previously described (15, 19). Mice were anesthetized by intramuscular injection of ketamine hydrochloride (100 mg/kg) and acepromazine maleate (5 mg/kg). The tracheas were surgically exposed, and 2.3 µl/g body wt of S. pneumoniae (5 × 109 CFU/ml) or E. coli (107 CFU/ml) were instilled intratracheally. All bacterial suspensions contained 5% colloidal carbon to mark the deposition of the instillate. Radiotracers for measurement of edema formation (see below) were injected into the tail vein 15 min before intratracheal instillation (125I-albumin) and 2 min before euthanasia (51Cr-RBC). After 6 h of infection, mice were killed by overdose of halothane. The peritoneal and thoracic cavities were rapidly opened, the heart vessels were tied off to prevent pulmonary blood loss, peripheral blood samples were drawn from the inferior vena cava, and the lungs were removed and fixed via intratracheal instillation of 6% glutaraldehyde under 22 cm H2O pressure. Circulating leukocytes were counted as above.
Pulmonary neutrophils were quantified by morphometry in histological sections (18). Carbon black-containing lung regions were embedded in paraffin, and 5-7 µm thick sections were cut and stained with hematoxylin and eosin. A counting grid (10 × 10, covering 70,000 µm2 of the magnified field) was reflected onto the field of view using a drawing tube. Randomly selected fields of pneumonic peripheral lung that were largely free of noncapillary blood vessels and bronchioles or larger airways were examined. A total of three grids (300 points) were counted for each lung, and every point was classified as landing on (a) air space or tissue and (b) neutrophil or not a neutrophil. The quantities of neutrophils in air space or tissue were expressed as volume % of the respective compartment (alveolar air space or septal tissue). The volume % of the alveolated region of lung occupied by air space and by tissue did not differ among infected and uninfected WT and CD18Peritonitis. Peritonitis was induced and characterized as previously described (15, 16). After anesthetizing mice by intramuscular injection of ketamine hydrochloride (100 mg/kg) and acepromazine maleate (5 mg/kg), iodinated albumin (0.3 µCi/mouse) was injected into the tail vein. S. pneumoniae (109 CFU/mouse) was injected intraperitoneally 15 min later. Mice were killed by a lethal overdose of halothane after 4 or 24 h of peritonitis, and the peritoneal cavities were lavaged with three aliquots of 5 ml of PBS. Peripheral blood was drawn from the inferior vena cava. Concentrations of total leukocytes in blood and lavage fluid were calculated from hemacytometer counts, and cellular differentials were quantified in smear or cytospin preparations stained with LeukoStat (Fisher Scientific Co.). The 125I-albumin recovered by lavage was expressed as the percentage of 125I-albumin injected and reflects plasma leakage into the peritoneal cavity (15, 16). Bacterial clearance was inferred from the loss of viable bacteria recovered in peritoneal lavage, and CFU recovered 4 or 24 h after instillation were expressed as a percentage of the original number of CFU injected. Sterile peritonitis was induced in separate groups of mice by intraperitoneal injection of 1 ml of sterile-filtered 4% thioglycollate in PBS, and mice were processed as above.
Statistics.
Groups consisted of four or five mice. Data were
presented as mean ± SEM. Data from different groups (WT versus CD18/
or infected versus uninfected) were compared by t
test. Because circulating blood cell counts in CD18
/
mutant
mice were not normally distributed and were highly variable, these data were compared by Mann-Whitney U test and expressed as medians in addition to mean ± SEM values. Differences were considered statistically significant when p <0.05.
Circulating neutrophils from WT mice expressed both CD11a
and CD11b on their surface, as measured by flow cytometry (Fig. 1). Neutrophils from CD18/
mutants, however,
did not express CD11a or CD11b (Fig. 1). Similarly, leukocytes from peripheral lymph nodes or spleens of WT
mice expressed CD11a and/or CD11b, whereas lymph
node and spleen cells from CD18
/
mutant mice did not
express CD11a or CD11b, as measured by flow cytometry
(data not shown).
Expression of CD11/CD18 by leukocytes marginated
within lung capillaries was examined by immunohistochemistry. Lungs from WT mice contained leukocytes
positive for CD11a or CD11b, but no leukocytes in
CD18/
lungs were positive for CD11a or CD11b. The
genetic mutation of CD18 resulted in the complete loss of
immunologically recognizable CD11a and CD11b in leukocytes of CD18
/
mutant mice.
All CD18/
mice with
or without experimentally induced inflammations had elevated circulating leukocyte and neutrophil counts when
compared with WT (Table 1).
|
CD18/
mice exhibited splenomegaly (spleen weights
of 171.7 ± 11.2 and 62.3 ± 5.3 mg in CD18
/
and WT
mice with bacterial peritonitis, respectively; p <0.05). Neither liver weights (1.02 ± 0.04 and 0.94 ± 0.04 g in
CD18
/
and WT mice, respectively) nor body weights
(18.9 ± 0.6 and 19.2 ± 0.8 g in CD18
/
and WT mice,
respectively) differed between WT and mutant mice with
bacterial peritonitis.
Croton oil application induced significant neutrophil emigration in WT
mice (Fig. 2). In contrast, there was no increase in emigrated neutrophils in the dermis of CD18/
mutant mice
after 6 h of croton oil dermatitis (Fig. 2).
Dermal Edema.
After 6 h, croton oil induced edema accumulation in both WT and CD18/
ears (Table 2).
There was, however, significantly less edema accumulation
in the CD18
/
ears than in WT ears (Table 2).
|
More neutrophils
were present in the alveolar septae of uninfected CD18/
mutants than in those of uninfected WT mice (Fig. 3).
There were almost no neutrophils in the alveolar air spaces
of WT and CD18
/
mutant mice (Fig. 4).
During E. coli and S. pneumoniae pneumonias, the volume fraction of septal tissue occupied by neutrophils increased in both WT and CD18/
mutant mice (Fig. 3).
CD18
/
mutants had more neutrophils in alveolar septae
for each type of pneumonia than did WT mice (Fig. 3).
Neutrophils emigrated into the alveolar air spaces during
E. coli and S. pneumoniae pneumonias for both WT and
CD18/
mutant mice (Fig. 4). During either pneumonia,
CD18
/
mutants had more neutrophils in their air spaces
than did WT mice (Fig. 4). The two types of bacteria induced a similar degree of neutrophil emigration in WT
mice, but fewer neutrophils (P <0.05) emigrated during E. coli than during S. pneumoniae pneumonia in CD18
/
mutant mice (Fig. 4).
The ratio of alveolar air space neutrophils to septal neutrophils in pneumonic regions is an index of the ability of
neutrophils to extravasate. In WT mice, the ratio of air
space to septal neutrophils was 1.0 ± 0.2 and 1.1 ± 0.1 during E. coli and S. pneumoniae pneumonias, respectively.
In CD18/
mutants, this ratio was 1.7 ± 0.2 and 2.3 ± 0.3 for E. coli and S. pneumoniae, respectively, significantly
greater than WT for each type of pneumonia (P <0.05).
Pulmonary edema during E. coli
pneumonia did not differ between WT and CD18/
mice
(Table 2). Edema accumulation was significantly greater in
CD18
/
mutants than in WT mice during S. pneumoniae
pneumonia (Table 2).
There were few
neutrophils in the uninfected peritoneal cavities of WT and
CD18/
mice (Fig. 5). After intraperitoneal injection of
S. pneumoniae, neutrophil accumulation was apparent 4 h
later, and was greatly increased after 24 h of peritonitis in
both WT and CD18
/
mutant mice (Fig. 5). More neutrophils were recovered in the peritoneal lavage fluids of
CD18
/
mutants than of WT mice after 4 or 24 h of peritonitis (220 ± 30 and 500 ± 50% WT, respectively; Fig.
5). After intraperitoneal injection of thioglycollate, there
was no significant difference between CD18
/
mutants
and WT mice in the number of neutrophils recovered by peritoneal lavage after 4 h (110 ± 30% WT; Fig. 6). By 24 h,
more neutrophils were recovered from CD18
/
mutants
than from WT mice (360 ± 80% WT; Fig. 6).
Peritoneal Edema.
Peritoneal edema did not significantly
differ between mutant and WT mice after 4 h of bacterial
or thioglycollate peritonitis (Table 2). After 24 h of either
peritonitis, there was more peritoneal edema in CD18/
mutants than in WT mice (Table 2).
Bacterial clearance
did not significantly differ between WT and CD18/
mutant mice. After 4 h, 24 ± 10% of injected CFU were recovered in the peritoneal lavage from WT mice, and 45 ± 9% of injected CFU from CD18
/
mice. After 24 h, <0.1%
of injected CFU were recovered from either WT or
CD18
/
mice.
Neutrophil emigration was reduced and edema accumulation compromised in CD18/
mutant mice with croton
oil dermatitis. In contrast, neither neutrophil emigration
nor edema accumulation were compromised in CD18
/
mutants during S. pneumoniae or thioglycollate peritonitis
or during E. coli or S. pneumoniae pneumonia. These data
confirm that CD11/CD18 complexes are essential to neutrophil emigration under at least some circumstances, but
they definitively demonstrate that CD11/CD18-independent pathways for neutrophil emigration can be used during acute inflammation in the peritoneum and the lung.
These results may be compared and contrasted with (a) studies using blocking antibodies to inhibit CD11/CD18 interactions with ligands, (b) observations with human LAD-1 patients, (c) studies with CD18 hypomorphic mutant mice with decreased but not eliminated expression of CD18, and (d) mutant mice deficient in CD11a or in CD11b.
Antibodies against CD18 inhibit cutaneous emigration
of neutrophils in response to diverse stimuli (2), and in this
study cutaneous emigration of neutrophils was inhibited
in CD18/
mutant mice during croton oil dermatitis.
Whereas antibodies against CD18 inhibit acute peritoneal
emigration of neutrophils during S. pneumoniae or thioglycollate peritonitis (3, 20) and acute pulmonary emigration
of neutrophils during E. coli pneumonia (3), however, neutrophil emigration was not inhibited during any of these
inflammatory reactions in mice completely deficient in
CD11/CD18 adhesion complexes due to a CD18 null mutation. This discrepancy suggests that the murine regulation
of adhesion pathways differs during lifelong deficiency of
CD11/CD18 compared with acute inhibition of CD11/
CD18 by antibodies.
Tissues from human LAD-1 patients with a lifelong deficiency of CD11/CD18 complexes are generally lacking in
neutrophils, even when infected by bacteria or fungi. Biopsies from diseased periodontal, gingival, pharyngeal-glottic,
dermal, or umbilical tissues from LAD-1 patients show a
lack of neutrophils in infected or inflamed sites (4, 5). Autopsy of a LAD-1 patient revealed infection without neutrophil emigration in the peritoneum, larynx, and esophagus (8). The lungs of the autopsied patient were also
infected, but in contrast to the other sites examined, emigrated neutrophils were observed (8). Thus, humans can
recruit neutrophils via CD11/CD18-independent pathways under at least some circumstances. During acute inflammations in this study, the CD18/
mutant mice recruited neutrophils into the lungs and the peritoneum, but
not into the skin.
Mutant mice that express 2-16% of WT levels of CD11/
CD18 on their leukocyte surfaces (CD18 hypomorphs)
have been previously characterized (13). The numbers of
neutrophils recovered by peritoneal lavage 4 h after intraperitoneal injection of thioglycollate were not significantly
different in CD18 hypomorphs when compared with WT
mice (13). Thus, either the remaining CD11/CD18 mediates neutrophil emigration in the hypomorphs, or alternative pathways for neutrophil emigration are used in the hypomorphs as in the CD18/
mutants.
Mutant mice lacking CD11a-CD18 secondary to a
CD11a mutation have been generated, and their neutrophils are compromised in their ability to emigrate during
thioglycollate peritonitis (21). In contrast, CD18/
mutants lack CD11a-CD18 as well as the other members of
the CD11/CD18 family, yet neutrophil emigration is not
compromised in these mice during peritonitis. Thus, the
CD18
/
mutants, but not the CD11a-CD18 mutants, induce CD11/CD18-independent neutrophil emigration during thioglycollate peritonitis. Mutant mice deficient in CD11b
do not demonstrate reduced neutrophil emigration during
thioglycollate peritonitis (22, 23). CD11a-CD18 function,
however, remains critical to neutrophil emigration in CD11b-deficient mice as indicated by CD11a-blocking antibodies (23). It is unclear why mutant mice with single deficiencies in CD11a-CD18 or CD11b-CD18 require CD11a-CD18 for
neutrophil emigration under circumstances in which CD18
/
mutants do not demonstrate impaired emigration. CD18
/
mutants are deficient in CD11a-CD18, CD11b-CD18,
CD11c-CD18, and CD11d-CD18, whereas the CD11a
/
and CD11b
/
mutant mice each express three of the four
adhesion complexes. Furthermore, the CD18
/
mutants
have extremely high circulating neutrophil counts, which has not been reported for the CD11a
/
or CD11b
/
mutants. These factors may contribute to the differing recruitment of CD11a-independent pathways for the emigration
of neutrophils in the peritoneal cavities of these mutant
mice.
To our knowledge, this is the first evidence that a
CD11/CD18-independent pathway can be used by neutrophils in nonpulmonary tissues during the first hours of
acute inflammation. CD11/CD18-independent neutrophil emigration in the peritoneum has been observed over prolonged (12-24-h) periods of inflammation (20, 24).
Macrophages recovered after 72 h of protease peptone-induced peritonitis can elicit CD11/CD18-independent
neutrophil emigration when transferred to naïve peritoneal
cavities before eliciting acute (4 h) streptococcal peritonitis
(25). CD11/CD18-independent neutrophil emigration also
occurs in the joints during chronic inflammation (26). In
CD18/
mutants, the peritoneal emigration of neutrophils
occurred in the absence of CD11/CD18 after only 4 h of
peritonitis.
These data suggest that neutrophils can use different adhesion molecule pathways in emigrating from the systemic
vasculature in the skin (during croton oil dermatitis) and in
the peritoneum (during S. pneumoniae or thioglycollate
peritonitis). These tissues may regulate the emigration of
neutrophils differently. Whereas neutrophil emigration into
the skin was quantified in situ by morphometric analysis of
histologic sections, neutrophil emigration into the peritoneum was quantified in lavage fluid. Studying the roles of
adhesion molecules in neutrophil emigration by examination of lavage fluid during peritonitis is complicated by the
fact that leukocytes use adhesion molecules, including
CD11/CD18 and ICAM-1, to adhere to the peritoneal
mesothelium (27, 28). Thus, leukocytes may be less likely
to adhere to the mesothelium and may be more likely to be
lavaged in situations in which CD11/CD18-ICAM-1 interactions are disrupted. Despite these confounding factors in interpreting lavage data, it remains clear that neutrophils emigrated during peritonitis in CD18/
mutant mice.
Neutrophil emigration was observed in situ during both E. coli and S. pneumoniae pneumonias in CD18
/
mutant
mice as well.
The observation of increased emigration of neutrophils
in the lungs and peritoneal cavities of CD18/
mutants
when compared with WT mice likely resulted from the increased numbers of circulating neutrophils in CD18
/
mutant mice, although other explanations such as decreased
resolution of inflammation (22) are also possible. Animals
rendered neutrophilic by administration of G-CSF demonstrate increased neutrophil numbers in bronchoalveolar lavage fluid during pneumonia induced by S. pneumoniae,
Klebsiella pneumoniae, or E. coli (29), and increased neutrophil numbers in peritoneal lavage fluid during peritonitis induced by Listeria monocytogenes, fecal flora, or E. coli (32- 34). The number of circulating neutrophils in CD18
/
mutants with pneumonia or peritonitis was increased 8-fold
or more when compared with WT mice. Thus, the increased neutrophil emigration observed in CD18
/
mutants with pneumonia or peritonitis likely resulted from
their peripheral neutrophilia.
In conclusion, CD18/
mutant mice displayed the expected phenotype of reduced acute neutrophil emigration
when studied during croton oil dermatitis. CD18
/
mutants, however, deviated surprisingly from what was expected when studied during acute S. pneumoniae or thioglycollate peritonitis or during acute E. coli pneumonia, in that
neutrophil emigration was not reduced, but was in fact increased during these types of inflammation. These genetically engineered mice now highlight and help to define
two important questions to be studied: (a) which signals induce neutrophils to emigrate via the CD11/CD18-dependent or -independent pathways?, and (b) which adhesion
molecules mediate CD11/CD18-independent neutrophil
emigration?
Address correspondence to Claire M. Doerschuk, Physiology Program, Harvard School of Public Health, Building I Room 305, 665 Huntington Ave., Boston, MA 02115. Phone: 617-432-1706; FAX: 617-432-3468; E-mail: cdoersch{at}hsph.harvard.edu
Received for publication 6 June 1997 and in revised form 7 August 1997.
1 Abbreviations used in this paper: LAD-1, leukocyte adhesion deficiency type 1; WT, wild-type.The authors thank Ervin Melulini for preparation of histological slides, and Amy Imrich for assistance with flow cytometry.
This work was supported by United States Public Health Service grants HL 48160, HL 52466, and AI 32177. Karin Scharffetter-Kochanek was supported by a Heisenberg grant from the Deutsche Forschungsgemeinschaft.
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