Immunopathology and Pulmonary and Critical Care Units, Massachusetts General Hospital, Boston, Massachusetts 02114
Submitted 26 July 2002 ; accepted in final form 6 March 2003
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ABSTRACT |
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lung; vessels; airways; immunity; lymphocytes
The activities of Epi are systemic and include the ability to modulate
immunity. Epi induces the release of lymphocytes from the murine spleen and
promotes their translocation into blood and lung
(14). Epi rapidly increases
circulating natural killer (NK) cells
(24), modulates T-helper (Th)
1 activities (7), mobilizes
granulocytes from marginal pools
(1), and increases
L-selectin expression
(20) and LPS-mediated
upregulation of TNF- receptors by blood monocytes
(11). In rats, Epi
administered daily before antigen sensitization and through subsequent antigen
challenge inhibits cutaneous tuberculin reactions
(12).
In the present study, we examined the effects of Epi on the pulmonary cell-mediated immune response to hen-egg lysozyme (HEL). HEL is a 14-kDa protein with five distinct T-cell epitopes. H-2k strains of mice show strong cell and antibody immune responses to HEL, whereas H-2b strains are weak responders (13). In a previous study, we demonstrated that lymph node cells from C57BL/6 (H-2b) mice immunized with HEL and injected daily with Epi in vivo show increased proliferative responses to HEL and increased IL-2 secretion in vitro compared with controls (21).
Cell-mediated pulmonary immune responses include afferent and efferent phases (28). During the afferent phase, antigen is taken up by antigen-presenting cells and transported from peripheral tissues to regional lymph nodes, where it is presented to naïve T lymphocytes. Upon subsequent intratracheal antigen challenge, sensitized immune cells accumulate around the pulmonary microvasculature and to a lesser extent around small pulmonary airways. In the present study, we examined whether Epi might promote the pulmonary cell-mediated immune response by C57BL/6 mice to HEL in vivo and yield differential responses when administered during either the sensitization or effector phases of the immune response.
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MATERIALS AND METHODS |
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Complete media and culture conditions. Cells were cultured in RPMI 1640 with L-glutamine (Mediatech, Herndon, VA), 10% heat-inactivated fetal bovine serum (Sigma, St. Louis, MO), 50 µg/ml gentamicin (GIBCO-BRL, Gaithersburg, MD), 0.5% 1 M HEPES buffer (GIBCO-BRL), and 2-mercaptoethanol (5 x 10-5 M; Sigma), and incubated at 37°C in a humidified chamber of 95% air and 5% CO2.
HEL immunization and challenge. Mice were immunized subcutaneously at both sides of the tail base on day 0 with HEL (200 µg; Sigma RBI) in complete Freund's adjuvant. On day 12, they were anesthetized with 4% chloral hydrate (400 mg/kg ip; Fisher Scientific, Pittsburgh, PA). The trachea was surgically exposed, and 200 µg of HEL in 100 µl of sterile normal 0.9% saline (Sal) were injected intratracheally. On day 15, mice were anesthetized with chloral hydrate and killed by cardiac puncture.
Epi and AR blocker administration. Mice were injected with Epi
(0.5 mg/kg; Abbot Laboratories; North Chicago, IL) or Sal subcutaneously on
days 17 [sensitization phase (SP)] or days
1214 [effector phase (EP)]. In some experiments, mice were
injected intraperitoneally 30 min before Epi with /
-AR blockers
including propranolol (10 mg/kg; Ben Venue Labs, Bedford, OH), phentolamine
(10 mg/kg; Ben Venue Labs), atenolol (10 mg/kg; Sigma), or ICI-118551 (10
mg/kg; Sigma). Controls received Sal injections intraperitoneally 30 min
before Epi.
Bronchoalveolar lavage. Bronchoalveolar lavage (BAL) was conducted as previously reported (28). Briefly, mice were anesthetized with chloral hydrate, the abdomen was surgically opened, and an incision was made in the diaphragm. The trachea was cannulated with polyethylene tubing and the lungs were lavaged with six to eight 1.0-ml aliquots of normal Sal containing 0.6 mM EDTA. The retrieved BAL cells were washed once with PBS, pH 7.3; hypotonic saline (0.2%) was added to the pellet to lyse red blood cells. Viable cells were enumerated in a hemocytometer by trypan blue exclusion, and cells were washed in PBS, pH 7.3, and resuspended in PBS with 0.5% BSA (Sigma) for immunostaining.
Immunostaining and cytofluorimetry of BAL leukocytes. BAL cells were incubated with Fc block (Pharmingen, San Diego, CA) for 10 min and stained in suspension for 30 min at 4°C with conjugated anti-mouse monoclonal antibodies. The fluorochrome-conjugated antibodies (all Pharmingen) included FITC- or phycoerythrin (PE)-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD25, anti-NK1.1, anti-class II major histocompatibility complex (Ia), and anti-CD11b. Anti-IgG2a-FITC and anti-IgG1-PE were used as fluorescence controls. Cells were washed in PBS and fixed in PBS with 1% paraformaldehyde (Fisher Biotech, Fairlawn, MO). We conducted dual color analysis of the cell surface membrane phenotype in a FACScan cytofluorimeter (Becton-Dickinson, Burlingame, CA) after electronically gating on the lymphocyte, monocyte, or granulocyte populations, as judged by forward-angle (0°) and side-angle (90°) light scatter characteristics.
Pulmonary cytokine gene expression. Cytokine and chemokine gene expression was determined by RNase protection assay (RPA). Total cellular RNA was extracted from lung digests with TRIzol (GIBCO Life Technologies, Gaithersburg, MD). RNA was assayed by absorbance at optical density at 260 nM (OD260), and RNA integrity was assessed by the OD260/280 ratio and by direct examination of 28S and 18S bands in 1% agarose gels. RPA was conducted with the RiboQuant Multiprobe RPA system kit (Pharmingen) according to the instruction manual. In brief, a 32P-labeled anti-sense probe transcribed from a multitemplate cDNA plasmid insert using T7 RNA polymerase was hybridized in excess with target RNA. The protected probe/RNA hybrids were treated with RNase to remove any remaining single-stranded probe and RNA, purified by standard chloroform/phenol extraction and ethanol precipitation techniques, and resolved on 5% denaturing polyacrylamide gels for imaging and quantification by autoradiography and phosphorimaging (Molecular Imager system; Bio-Rad Laboratories, Hercules, CA). GAPDH served as an internal standard for each sample.
Histochemistry. Lungs were harvested, fixed in 10% buffered formalin, sectioned at 5 µm, and stained with hematoxylin and eosin. Airways and vessels were scored for intensity of inflammation in five randomly selected high-power fields under a light microscope. The scoring system was as follows: vessels: 0 = no inflammation, 1 = incomplete leukocyte perivascular cuff, 2 = complete leukocyte perivascular cuff up to two cell layers thick, 3 = complete perivascular leukocyte cuff greater than two cell layers thick; airways: 0 = no inflammation, 1 = peribronchial leukocyte cuff less than two cell layers thick, 2 = peribronchial leukocyte cuff 24 cell layers thick, 3 = peribronchial leukocyte cuff greater than four cell layers thick.
Immunohistochemistry. Lungs were rapidly frozen in cryoembedding medium, sectioned at 5 µm in a cryostat and stained with rat anti-mouse monoclonal antibodies [anti-CD3, anti-CD11b, anti-NK1.1, anti-GR-1, anti-CD31, anti-intracellular adhesion molecule (ICAM)-1; all Pharmingen] by an avidin-biotin immunoperoxidase technique, as previously described (26). Positive cells were scored with a Zeiss light microscope in a minimum of five random high-power (x25 objective) fields.
Electron microscopy. For ultrastructural analysis, lung tissue was finely sectioned and fixed for 3 h at room temperature in modified Karnovsky's solution (2.5% glutaraldehyde, 2.0% formaldehyde, and 0.0025% CaCl in 0.1 M sodium cacodylate buffer, pH 7.4), transferred to cacodylate buffer, and stored at 4°C until ready for processing. Subsequently, tissue was processed in a Leica Lynx EM tissue processor, postfixed in osmium tetroxide, stained en bloc with uranyl acetate, dehydrated in graded ethanol solutions, infiltrated with propylene oxide/epon, and embedded in epon. Tissue was sectioned at 1 µm, stained with toluidine blue, and examined by light microscopy. Representative areas were chosen, and thin sections were cut with a diamond knife in an LKB Ultrotome III ultramicrotome. Sections were stained with Sato's lead citrate and examined in a Philips 301 electron microscope.
Cortisone assay. Blood was collected at the time of death by cardiac puncture. Plasma was retrieved and frozen at -80°C until time of assay. Corticosterone was assayed by competitive RIA kit (ICN Pharmaceuticals, Costa Mesa, CA).
Statistical analysis. Data were analyzed by paired or unpaired t-statistics or ANOVA using Statview 4.5 software.
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RESULTS |
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To ascertain the specificity of the response to EPI, mice were treated
concomitantly with /
-AR blockers
(Table 1). Vascular
inflammation in situ was reversed by atenolol (P < 0.05), a
selective
1-AR antagonist, but not by ICI-118551, a selective
2-adrenoreceptor antagonist, propranolol, or phentolamine
(Fig. 3).
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Epi-SP mice showed increased immune cell cuffing of airways
(Fig. 4). Airway inflammation
was reversed nonselectively by both /
-AR blockade (P
< 0.05).
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When Epi was administered during the effector phase (Epi-EP) at days 1214, airway inflammation was increased (P < 0.001), but there was no increase in vascular inflammation compared with controls (Fig. 5). These findings suggest that distinct AR-mediated mechanisms regulate the inflammation in pulmonary vessels and airways.
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Increased vascular inflammation in Epi-SP mice is characterized by the accumulation of GR-1+ granulocytes and CD11b+ monocytes. Immunostaining of lung sections revealed no significant increases in CD3+ lymphocytes, NK1.1+ lymphocytes, or CD31+ platelets in the inflamed vessels of Epi-SP mice compared with controls (not shown). GR-1 granulocytes (31 ± 6 vs. 20 ± 5, P = 0.0001) and CD11b+ macrophages (76 ± 11 vs. 45 ± 10, P < 0.0001) were both increased in perivascular cuffs compared with controls (Fig. 6).
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Epi induces changes in lymphocyte subsets in the BAL fluid. Neither Epi-SP- nor Epi-EP-treated mice showed changes in the total number of leukocytes in bronchoalveolar lavage fluid (BALF), and there were no differences in the percentages of total lymphocytes, macrophages, or granulocytes compared with controls (data not shown).
Cytofluorimetric analysis demonstrated a modest increase in the percentage
of CD3+ lymphocytes (Table
2) in Epi-SP mice (P < 0.05), and this change was
reversed by propranolol (P < 0.05). Epi-SP decreased the
percentage of NK1.1+ cells by 50% (P < 0.01). This
finding was reversed by both phentolamine and propranolol (P <
0.05, P < 0.01) but not by selective
-AR blockade,
suggesting a requirement for the cooperation of both
1- and
2-AR in this response. Epi-SP yielded no change in either the
percentages or absolute numbers of CD4+, CD8+, or
CD19+ lymphocytes in the BALF (not shown).
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Epi-SP reduced both the percentage and total number of
CD4+CD25+ lymphocytes in BALF by 50% (P
< 0.05) (Fig. 7). This
finding was selectively reversed by the
1-AR blocker atenolol
(P < 0.05) but not by ICI-118551, phentolamine, or
propranolol.
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Epi-SP increased the percentage of CD11b+ monocytes in BALF from
8.4 ± 5.7 to 15.7 ± 9.8 (P < 0.05) paralleling their
increased representation in perivascular cuffs within the lung. However, this
finding was not reversed by either /
-AR blockade. No change was
observed in the representation of GR-1+ granulocytes in the BALF
compared with controls.
In contrast to the Epi-SP mice, the Epi-EP mice showed decreased percentages of CD3+ lymphocytes (73 ± 3 vs. 67 ± 1) and increased NK1.1+ cells (13 ± 3 vs. 18 ± 2) in the BALF (both P < 0.05).
Epi-SP has no effect on expression of pulmonary chemokine genes.
As chemokines expressed in lung tissue play a role in directed leukocyte
traffic, lung homogenates were assayed for expression of lymphotactin;
regulated on activation, normal T cell expressed, and presumably secreted;
eotaxin; macrophage inflammatory protein (MIP)-1; MIP-1
;
IFN-inducible protein-10; monocyte chemoattractant protein-1; and T cell
activated-3 mRNA by RPA. No differences were found between Epi-SP and Sal
groups (not shown).
Epi-EP yields no difference in cortisone levels in the efferent
response to HEL. Cytokines including IL-1, TNF-
, and IL-6
can stimulate both sympathetic arousal and the hypothalamic-pituitary adrenal
axis by activating release of corticotropin releasing factor
(6). To exclude the possibility
that Epi-SP might alter pulmonary cell-mediated immunity by modulating
cortisone (Cort) release, plasma Cort was assayed at the time of death. There
was no difference in plasma Cort levels between Epi-SP and Sal controls (180
± 105 vs. 207 ± 99, P = 0.6).
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DISCUSSION |
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Efforts to determine the cellular characteristics of this response showed
that Epi yielded modest but significantly increased percentages of
CD3+ T lymphocytes and concomitantly decreased NK1.1+
lymphocytes in the BALF. These effects were differentially mediated by
- and
-AR. Whereas propranolol, a nonselective
-AR blocker,
reversed the increase in CD3+ lymphocytes, the decrease in NK cells
was reversed by both propranolol and phentolamine, suggesting a role for both
/
-AR regulation. The effects of
-AR in the reduction of NK
cells appeared to require the cooperation of both
1-AR and
2-AR, because neither atenolol nor ICI-118551 alone yielded
this effect. Administration of both Epi and atenolol, a selective
1-AR blocker, decreased NK cells below levels observed with
Epi alone. This suggests that
1-AR may regulate NK cell
motility in the context of increased adrenergic tone. Neither propranolol nor
phentolamine diminished the increased vascular inflammation yielded by Epi-SP,
suggesting that this change was not directly related to the representation of
CD3+ and NK1.1+ lymphocytes in the BALF.
Epi-SP yielded decreased percentages of CD4+ CD25+
lymphocytes in BALF, and this was specifically reversed by atenolol, a
selective 1-AR blocker. Because vascular inflammation was
also specifically reversed by atenolol, CD4+CD25+
lymphocytes may play a direct role in this response. Propranolol, a
nonselective
-AR blocker, did not reverse the decline in
CD4+CD25+ lymphocytes or vascular inflammation. This may
be due to differential effects of selective
1-AR blockade vs.
blockade of both
1-AR and
2-AR. No
conclusive role for either receptor subtype in vascular inflammation has
previously been described.
The changes produced by Epi-EP were distinct from those observed in the SP. Whereas Epi yielded increased airway inflammation in both phases, vascular inflammation was not increased above controls in response to Epi-EP. Furthermore, Epi-EP decreased the percentage of CD3+ lymphocytes but increased the percentage of NK1.1+ cells in the BALF, in contrast to Epi-SP.
Both norepinephrine and Epi mediate their effects on immune cells by
stimulating - and
-AR. At least three subtypes of
-AR
(
1,
2,
3) and two
-AR (
1,
2) have been identified
(8). Lymphoid cells express
-AR, and both T and NK lymphocytes express
2-AR. Maisel
and colleagues (17) have
suggested that
2-AR density differs among leukocytes with NK
cells > CD14 monocytes > T cytotoxic cells > Th cells. Sanders and
coworkers (23) have
demonstrated that unlike Th1 lymphocytes, Th2 lymphocytes do not express
2-AR. However,
2-AR are not hypothesized to
play a critical role in the observed vascular inflammation because ICI-118551,
a selective
2-AR blocker, did not reverse it. Although
1-AR appear to play a critical role in Epi-induced pulmonary
vascular inflammation, the representation of
1-AR on
leukocytes is controversial
(1).
The mechanisms by which -AR modulate airway inflammation and
trafficking of NK1.1+ cells induced by Epi-SP are uncertain.
Increased granulocyte release from marginal pools in bone marrow, blood
vessels, and lung is mediated by
2-AR
(1). Platelets also express
2-AR but the expression of
-AR by monocytes and
lymphocytes has not been convincingly established
(2).
Reduced representation of CD4+CD25+ lymphocytes in
BALF was a consistent finding in the response to Epi-SP.
CD4+CD25+ lymphocytes include a quantitatively small
immunoregulatory cell subset that has been demonstrated to prevent the
induction of a variety of autoimmune diseases in mice, including gastritis,
insulitis, adrenalitis, and polyarthritis
(22). Decreasing the frequency
of CD4+CD25+ cells in murine pancreatic lymph nodes
yields greater induction of autoimmune diabetes
(9). As vascular inflammation
is a feature of most autoimmune disorders, the reduction in
CD4+CD25+ lymphocytes may contribute to heightened
vascular injury via as yet uncertain mechanisms. Currently, no information
exists concerning the expression of AR by the CD4+CD25+
lymphoid subset. However, the ability to modulate the representation of
CD4+CD25+ lymphocytes with atenolol suggests that these
cells may be distinguished by their expression of 1-AR.
The acute administration of catecholamines in murine species yields a
transient increase in circulating lymphocytes with small increases in
CD3+ and CD8+ lymphocytes and large increases in NK
cells (1,
24). But repeated challenges
with the 2-AR agonist terbutaline yields decreased numbers of
circulating NK lymphocytes without significant changes in CD3+
lymphocytes (18). The
mechanisms underlying the difference in the acute and chronic effects of
-adrenergic stimulation have not been determined but may include the
acute mobilization of NK cell depots in the spleen and the marginating blood
pool (24) that are exhausted
by prolonged
-adrenergic administration. In the present study, the
larger total dosage and duration of Epi administration to Epi-SP mice may
explain why NK cells were decreased compared with Epi-EP mice. However, the
absence of correlation between NK1.1+ lymphocyte representation and
vascular inflammation suggests that NK cells are not the primary mediator of
the Epi-heightened immune response in this model.
Catecholamines can inhibit Th1 responses and favor the development of Th2
responses (7). -AR
stimulation inhibits IL-12 production by monocytes and dendritic cells
(27) and blocks TNF-
release by LPS-treated monocytes and glial cells in vitro. However, the
effects of catecholamines on inflammation may be organ specific and
compartmentalized. For example,
2-AR stimulation augments
LPS-stimulated release of TNF-
by peritoneal macrophages
(25) and increases expression
of TNF-
and IL-1 by lung macrophages.
-AR also potentiate the
release of IL-8 from human monocytes and lung epithelial cells
(15), effects that would be
expected to augment pulmonary Th1 inflammation. Some studies suggest that Th1
responses are increased by catecholamines. Dhabhar and McEwen
(5) demonstrated that stress
augments Th1 antigen-mediated cutaneous cellular immunity in vivo.
Catecholamines can modulate chemokine expression. As noted,
2-AR stimulation promotes expression of IL-8 by human lung
monocytes (15). Hasko et al.
(10) demonstrated that
MIP-1
, a proinflammatory chemokine that participates in the recruitment
of leukocytes to the lung, is downregulated by endogenous and exogenous
-AR stimulation. But in the present study, we were unable to detect
significant differences in the expression of MIP-1
and other
proinflammatory cytokines by RPA.
The increased pulmonary vascular inflammation observed in the present study
may be mediated primarily by nonimmune factors. Epi increases cardiac output
by promoting cardiac rate and myocardial contractility via
1-AR stimulation
(16). In addition, Epi also
vasoconstricts peripheral blood vessels. These hemodynamic effects can
potentially contribute to shear stress to the pulmonary endothelial lining
(3), which can injure
endothelial cells directly and/or upregulate their expression of
immunomodulatory adhesion molecules
(19), including ICAM or
vascular adhesion molecules. Increased adhesion molecule expression promotes
adherence of circulating leukocytes, including monocytes and granulocytes.
This could account for why the increased pulmonary vascular inflammation in
the present study was inhibited by atenolol, a
1-AR
antagonist. However, this does not fully explain why vascular injury was not
also inhibited by propranolol, a nonselective
-AR blocker.
We conclude that the effects of Epi on cell-mediated vascular and airway
inflammation are dissociable and may reflect selectively patterned
interactions with /
-receptors distributed in tissues. The present
findings suggest that Epi promotes vascular injury via
1-AR
mechanisms in vivo. They do not support a role for a
2-AR
mechanism. The findings in these studies raise potentially important questions
concerning the use of catecholamines in medical practice, particularly in
asthma and shock states where new exogenous or endogenous antigen challenges
may occur. We speculate that Epi-mediated microvascular injury is exacerbated
by pulmonary cell-mediated immunity. Future studies will examine the
expression of
/
-AR by CD4+CD25+
lymphocytes, the mechanisms of
1-AR-mediated vascular injury,
and AR-mediated airway inflammation.
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ACKNOWLEDGMENTS |
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This research was supported by National Institute of Allergy and Infectious Diseases Grant R01 AI-39054 and by the Mind/Body Medical Institute, Boston, MA.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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