* Asthma, Allergy, and Airway Research Center, Division of Pulmonary, Allergy, and Critical Care Medicine,
Department of Surgery, and
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pennsylvania
Correspondence: Bill T. Ameredes, Ph.D., Assistant Professor of Cell Biology and Physiology, Asthma, Allergy, and Airway Research Center, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh School of Medicine, 628NW Montefiore University Hospital, 3459 Fifth Ave., Pittsburgh, PA 15213. E-mail: ameredesbt{at}msx.upmc.edu
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3x104 cells) with the
exception of a doubling in the S/C IL-10-/- mice
(6x104 cells, P<0.05). NO production (per
million cells) was doubled in cells from S/C IL-10-/-
(15.3 µM) mice compared with WT (7.6 µM, P<0.05).
Inhibition of iNOS by
L-N5-(1-iminoethyl)-ornithine reduced NO
production in all S/C mice, confirming that the increase was a result
of up-regulation of iNOS. We conclude that IL-10 is a critical cytokine
regulating iNOS in murine airway cells and that its absence can lead to
up-regulation of iNOS and development of allergic airway
inflammation.
Key Words: allergen • bronchoalveolar lavage • C57BL6 mice • macrophage • nitrite • ovalbumin
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) [4
],
which have been triggered by the presence of allergens or
pro-inflammatory mediators. With production of IL-10, a negative
feedback loop allows down-regulation of TNF- [5
] and
subsequent resolution of the inflammatory response [6
].
This resolution may include the effect of IL-10 to inhibit eosinophil
survival and cytokine production [7
], which has
significant ramifications for diseases such as asthma, characterized by
immune cell infiltration into the airway. However, the ability of airway-immune cells to produce IL-10 has been shown to be reduced in asthmatics [8 , 9 ]. It is possible that lack of this anti-inflammatory cytokine may allow the pro-inflammatory mechanisms to dominate unchecked, resulting in persistent airway inflammation and possibly airway remodeling. An animal model of this situation is the transgenic IL-10 knockout (-/-) mouse, which has an inability to produce IL-10 [10 ] and is considered a good model in which to study inflammation [11 ]. We used this animal model to better understand the potential inflammatory mechanisms within the airways.
In addition to its anti-proliferative properties, IL-10 is known to suppress cellular production of nitric oxide (NO), a critical molecular signal in the inflammatory process, which is likely linked to asthma, airway inflammation, and possibly airway remodeling [12 , 13 ]. In the airway, inducible NO synthase (iNOS) can be up-regulated by pro-inflammatory cytokines and is considered a marker of airway inflammation in asthma [14 ]. IL-10 has been shown to reduce iNOS levels and NO production in a murine macrophage cell line [15 ], further supporting the idea that it has anti-inflammatory properties in airway-immune cells. However, in the presence of an allergen-potentiated inflammatory stimulus, the pattern of iNOS activity by airway-immune cells of the IL-10-/- mouse is unknown. Based on the known suppressive effect of IL-10 on iNOS induction and NO production [15 ], we postulated that iNOS activity and therefore NO production might be elevated in airway cells from mice lacking the ability to produce IL-10. The results are consistent with this postulate, suggesting that a lack of IL-10 may be an important factor in the development of asthma and airway inflammation through NO-dependent pathways.
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Allergen sensitization and airway challenge
Two groups of mice (WT and IL-10-/-,
n=16/group) comprised a set that was allergen-sensitized and
airway-challenged (S/C). Sensitization was achieved by using 0.5 ml
injections, interperitoneally (i.p.), of ovalbumin (50 µg/ml, Sigma
Chemical Co., St. Louis, MO; Grade VI) and alum (1 mg/ml) as a mild
adjuvant dissolved in normal saline. All mice were injected once/day
over a period of 7 days. This set of mice was subjected to a 10-min
airway-allergen challenge (48 h later), using ovalbumin/saline aerosol
(5 mg/ml) once/day over a period of 7 days. The aerosol was delivered
to eight unanesthetized mice simultaneously, using a DeVilbiss
nebulizer (particle size<3.5 µm) connected to a mixing chamber,
which in turn was connected to eight aerosolization chambers (Buxco
Electronics, Sharon, CT). This aggressive sensitization and challenge
protocol was purposely chosen to maximize eosinophilia in the airway
[16
] and the potential for inflammation-driven induction
of iNOS within the airway [17
]. Two other sets of WT and
IL-10-/- mice were also studied, those that were
unsensitized/unchallenged or naive (U/U, n=8/group) and
those that were subjected to the sensitization injections alone (S/U,
n=4/group).
Cell isolation and culture
Forty-eight hours after the last aerosol challenge, the mice
were anesthetized with Metofane, and open-chested bronchoalveolar
lavage (BAL) was performed using sterile, endotoxin-free, normal saline
to obtain airway cells from both lungs. Unfractionated BAL cells were
used to expedite processing and enhance viability. It also allowed the
airway cell population milieu to remain intact, providing a more
physiologic assay of cell signaling and cell contact interactions,
resultant of the allergic treatment regime. The cells were
immediately centrifuged (10°C, 450 x g), washed,
isolated, and placed in a 96-well polystyrene culture plate with
Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum,
L-glutamine, and penicillin/streptomycin. Media containing
added nitrate, such as RPMI-1640, was avoided to enhance the
probability that measured nitrite production was solely a result of
cellular activity [18
]. Cell culture plating density was
300,000–500,000 cells/well, in a total volume of 250 µL/well. The
plate was then placed in a humidified incubator (10% CO2
at 37°C) for 24 h. The conditioned media was collected after
24 h, snap-frozen (-80°C), and later assayed or assayed
immediately after collection. In the first set of experiments
(n=8 mice/group, WT and IL-10-/-,
respectively), conditioned media from unstimulated cells and cells
treated with lipopolysaccharide (LPS, 1000 ng/ml) and interferon gamma
(IFN-
, 100 U/ml) was collected. In the second set of experiments
(n=8 mice/group, WT and IL-10-/-,
respectively), stimulated and spontaneous production of nitrite was
measured after selective iNOS inhibition by
L-N5-(1-iminoethyl)-ornithine (L-NIO; 80 µM;
Sigma Chemical Co.) [19
]. All samples were assayed in
duplicate, and the mean of the duplicates was taken as the value for
that sample. A small aliquot of the original BAL sample was used to
determine the differential cell counts using Diff-Quik staining
(Scientific Products, McGaw Park, IL), and another aliquot was used for
cell viability, using the Trypan blue exclusion method. Typically,
200–300 cells were counted per slide to make these assessments. Cell
numbers were expressed per body weight of mouse to normalize for size
differences among mice.
Nitrite measurement
Nitrite production by cultured airway cells was measured as an
index of iNOS activity, using a standard Griess assay
[20
]. The resultant colorimetric reaction was assessed
using a plate reader that converted optical density values to
concentrations of nitrite. A standard curve was produced for known
nitrite concentrations from 0.5–20 µM, which was linear for
concentrations 
2 µM and for which the lower limit of reliable
detection fell between 1 and 2 µM. Therefore, sample values <2 µM
were considered to be beneath the limit of detectability of the assay.
Final nitrite values were adjusted to reflect nitrite production per 1
million cells. Nitrite content of cell-free culture medium with serum
added was always far below the level of detectability (typically 0–0.5
µM), suggesting that measurable nitrite production above the level of
detectability was a result of cellular activity as a function of the
treatment group [18
].
iNOS mRNA quantification
The quantification of iNOS mRNA was performed by Northern gel
analysis on RNA extracted from the lungs. Whole lungs were removed
immediately after BAL, placed in Ultraspec (Biotecx Laboratories,
Houston, TX), homogenized, and frozen at -80°C for later RNA
extraction. mRNA was extracted with chloroform, followed by
phosphate-buffered saline and isopropanol precipitation. The subsequent
pellet was washed twice with 70% ethanol/diethyl pyrocarbonate
water. The pellet was reconstituted in DEPC water, and the
concentration of mRNA was measured spectrophotometrically. A 10 µL
aliquot of the mRNA sample was loaded into a formaldehyde/agarose gel
with an ethidium bromide buffer. Separation of mRNA was achieved by
application of a current of 125V for 2.5 h. The gel was washed
with water to remove the formaldehyde and ethidium bromide, and the
mRNA was transferred with overnight incubation to a nylon membrane
(GeneScreen, NEN Lifescience, Boston, MA). The mRNA was
cross-linked to the membrane, hybridized for 2–4 h, and probed with an
iNOS-specific radioactive (32P) probe (cDNA iNOS probe, Alexis, San
Diego, CA) during a 24-h incubation. The membrane was washed,
blotted dry, and placed on a phosphor-image development cassette for
24 h. Subsequent quantification of bound radiation label was
provided by computerized phosphor-image analysis (Storm 2000,
ImageQuest, Molecular Devices, Palo Alto, CA), with resultant
densitometry values adjusted for 18S loading and expressed as
fractional increases with reference to control.
iNOS protein determination
Presence of iNOS protein within cultured BAL cells was
determined after cold, 70% ethanol-fixation onto glass slides and
subsequent staining using a primary rabbit anti-iNOS antibody coupled
to a flourescent goat anti-rabbit Alexa 488 secondary antibody
(Molecular Probes, Junction City, OR). Cells were observed at 40x
magnification under flourescent microscopy (Olympus BX51) with a
standard fluorescein isothiocyanate (FITC) crystal using a 488 nm
excitation wavelength. Images of the cells were obtained using software
(Optronics Magnafire, Spectra Services, Webster, NY) running on an
IBM-PC-compatible pentium computer.
Statistics
Spontaneous and stimulated cell values of nitrite were compared
within WT or IL-10-/- groups by using a nonparametric
Wilcoxon signed-rank test (SigmaStat, Jandel Corp., San
Rafael, CA) with values of P < 0.05 considered
significant. Nonparametric rank-sum tests were used to make all other
comparisons across groups and treatments with values of
P < 0.05 considered significant. Mean values for all
other groups that were beneath the limit of nitrite assay detectability
were graphically represented as "N.D." or had means shown if a
value for one or more of the individual samples exceeded the
detectability limit and remained within three standard deviations of
the mean of their respective groups.
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View this table: [in a new window] |
Table 1. BAL Cell Counts and Differentials of Cells Assessed for Nitrite
Production
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 View larger version (14K): [in a new window] |
Figure 1. Nitrite levels produced by airway immune cells from naive (U/U),
sensitized-only (S/U), and sensitized + airway-challenged (S/C) WT
(wild-type) and IL-10 knockout (-/-) mice. N.D. = not
detected;  , P < 0.05
IL-10-/- S/U compared with IL-10-/- naive;
+, P < 0.05 compared with respective S/C spontaneous
(spont) group; , P < 0.05 stimulated (stim) WT S/C
compared with respective stimulated S/U group; , P < 0.05 stimulated IL-10-/- S/C compared with respective
stimulated S/U and naive groups; *, P < 0.05
stimulated S/C IL-10-/- compared with stimulated S/C WT;
nitrite levels are expressed as values per 1 million cells.
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iNOS mRNA
iNOS mRNA was elevated in the lungs of the S/C
IL-10-/- mice compared with those of the U/U
IL-10-/- mice (Fig. 2
). Lane-loading of these samples was nearly identical, as indicated
by the 18S densitometry values; therefore, the darkness density
provides a good visual quantitation of the increase in mRNA in these
samples. Actual quantitation indicated that iNOS mRNA (25S) was
up-regulated by 76% in the sensitized and challenged
IL-10-/- mouse lungs.
 View larger version (49K): [in a new window] |
Figure 2. iNOS mRNA levels in naive (U/U) and sensitized and challenged (S/C)
IL-10-/- mouse lungs. Band densitometry values indicate
18S mRNA loading. Graph at bottom shows relative increase in iNOS mRNA,
as determined by fluorescence imaging densitometry.
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 View larger version (10K): [in a new window] |
Figure 3. iNOS protein in cultured, stimulated BAL cells from (A) naive
wild-type, (B) naive IL-10-/-, (C) sensitized +
airway-challenged WT, and (D) sensitized + airway-challenged
IL-10-/-. Faint to dark blue color indicates cell nuclei
as a result of secondary antibody stain; green is iNOS protein as a
result of anti-iNOS antibody stain. Original magnification =
40x.
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BAL differential cell-count shifts
The BAL differential cell counts displayed several important
properties in these experiments. One property was that eosinophil
numbers remained negligible with systemic allergen sensitization alone
(S/U) in WT and IL-10-/- mice (Table 1)
. This stability
suggested that the systemic sensitization alone did not potentiate
airway inflammation. However, with airway challenge after
sensitization, the eosinophil numbers rose significantly in both
groups. In conjunction with the eosinophil numeric stability shown in
the naive and S/U groups, this finding indicates that the
airway-inflammatory response was a result of the addition of
airway-allergen challenge in the setting of prior systemic allergen
sensitization. It should be noted that these eosinophil numbers were
not statistically different between the WT and IL-10-/-
BAL cells that were assessed for nitrite production in the present
study. This suggests that the eosinophilic recruitment mechanism
produced with airway inflammation is similar in mice congenic in the
C57BL6 background.
Another important property of the BAL differential cell counts was that
macrophage numbers remained similar across all treatments of the WT
mice (3x104 cells/g mouse) and were not different than
that of the naive or S/U IL-10-/- mice. This suggests
that significant numbers of macrophages were not recruited to the
airway with the airway challenge and subsequent inflammation in the WT
mice. However, with S/C treatment, the number of macrophages in the
IL-10-/- mice was nearly doubled (6x104
cells/g mouse), suggesting that macrophage recruitment is an important
part of experimental allergen-induced airway inflammation in the
absence of IL-10. Thus, this finding is relevant because increased
macrophage influx is a feature of human airway inflammation in response
to allergen challenge [21
]. The presence or addition of
IL-10 protein and the transfer of the IL-10 gene to the airway have
been shown to inhibit recruitment of airway-immune cells in murine
models of airway inflammation [6
, 22
].
Conversely, airway-immune cell recruitment has been shown to be
enhanced in IL-10-/- mice and in asthmatic humans in
which IL-10 production is suppressed in the setting of allergic airway
inflammation [3
, 21
]. Our findings are
similar to the latter and are consistent with the idea that a lack of
IL-10 results in increased infiltration of airway macrophages as a
result of airway inflammation.
iNOS induction
We observed that production of nitrite by airway cells was
significantly increased by sensitization and challenge in WT and
IL-10-/- mice, suggesting that such treatments induce
expression of iNOS. Further, enhanced production of nitrite in
IL-10-/- mice was more than double that seen in the WT
control mice, which suggests that endogenous IL-10 may down-regulate
expression of iNOS, as postulated by Warner and colleagues
[23
]. The L-NIO experiments indicate that increased
nitrite production was related to increased iNOS activity. These
findings were further supported by the fact that increased iNOS mRNA
and protein were observed in tissue and cells from the S/C
IL-10-/- mice.
It is likely that the source of nitrite production in the cell cultures
from S/C mice was the alveolar macrophages present in the airway-immune
cell isolate. This idea is supported by the fact that macrophages are
known to produce significant amounts of nitrite when stimulated by
IFN- and LPS [15
, 23
]. In all cases in
the present study, macrophages constituted the majority of the cells
obtained from the airways (up to 97%), even when significant numbers
of eosinophils were recruited as part of the inflammatory process. The
fact that macrophage numbers were only increased in the S/C
IL-10-/- mice is further suggestive that they were a
likely source of elevated nitrite production in this group.
Consideration of the fact that the nitrite levels were adjusted for
cell numbers across all samples and groups, this finding of macrophage
doubling and nitrite-output doubling is suggestive that the response to
inflammation in our model may be dependent on macrophage-activation
state in addition to macrophage-recruitment numbers in the absence of
IL-10. This possibility is consistent with evidence that alveolar
macrophages from asthmatics are activated in response to allergen
[24
], potentially supporting their role in allergic
airway inflammation and the production of NO.
Our findings of increased nitrite production by ex vivo BAL cells are consistent with the idea that allergic inflammation can potentiate cytokine-stimulated NO production in these cells. This may have relevance in the case of asthma, in which NO is considered to be a marker of airway inflammation [14 ], production of pro-inflammatory cytokines is associated with the allergic inflammatory response [4 ], and IL-10 production by recruited airway-immune cells is significantly diminished or absent [8 , 9 ]. A potential dilemma concerning inflammation-driven NO production in the airway stems from its potential to be a bronchodilator. However, we speculate that it is possible that NO may be serving as a cellular signal within the scheme of the inflammatory pathways, as opposed to a generic airway smooth-muscle relaxant under these circumstances. This notion would be consistent with its presence in allergic asthma [4 ], wherein increased bronchoconstriction is known to occur.
Critique of methods: BAL macrophages
Because of the limit of our experimental design, in which we
assayed production by all BAL cells collected, we do not know for
certain which cell types produced the elevations in nitrite and
exacerbations of inflammation that we observed. However, one strength
of our design was that the BAL isolate was likely representative of the
milieu of cells present in the airway under each experimental
condition. We have focused on the macrophage as the likely source cell
for iNOS up-regulation in the absence of IL-10 in our knockout model.
However, there are other studies that indicate some controversy. For
instance, NO production was shown to be increased as a function of
IL-10 dose in murine bone marrow-derived macrophage cell cultures
[25
], and IL-10 has been shown to only weakly inhibit
release of reactive nitrogen intermediates from murine peritoneal
macrophages [26
]. In general, the consistency of our
results with prior studies of iNOS up-regulation in alveolar
macrophages [15
, 23
] suggests that the
effect of IL-10, or its lack, is in part dependent on the source and
treatment of the macrophage cells. In addition, there has been a
historical divergence as to whether the classic up-regulation of iNOS
and NO production in mouse macrophages provides an adequate model of
mechanisms in human macrophages typically thought to be refractory to
this up-regulation [27
]. However, more recent studies
using improved and more sensitive techniques have indicated that
up-regulation of iNOS and NO production does occur in human
macrophages, particularly in certain disease states
[16
]. That airway NO levels are increased in asthma,
wherein IL-10 capacity of airway-immune cells is markedly compromised
[8
, 9
]. Similar to that seen in the present
study, macrophages aggressively infiltrate the segmentally
antigen-challenged human airway [21
]. These two facts
suggest that the IL-10-/- mouse model is likely relevant
to understanding mechanisms in airway inflammation.
Critique of methods: iNOS up-regulation
We measured nitrite levels as an indicator of NO production and as
an index of NOS induction and activity within the airway-immune cells
isolated by BAL. The relationship between nitrite production and NOS
content within mouse macrophages has been shown to be reasonably
consistent [15
]. Those findings, together with the
elimination of nitrite production by L-NIO, enhance our confidence that
the significantly elevated nitrite levels in the S/C
IL-10-/- cells were a result of enhanced induction of NOS
in the absence of IL-10. This supports the idea that the process of
inflammation was modulated differently in the IL-10-/-
mice, being exacerbated without the anti-inflammatory influence of
IL-10 present. However, another interpretation that cannot be ruled out
is that of enhanced sensitivity of the cells from the S/C mice to
IFN- and LPS stimulation as a result of airway inflammation in the
absence of IL-10. Further elucidation of the specifics of this
mechanism awaits subsequent study.
Summary
In summary, we show that in a murine model of allergic
sensitization and challenge, IL-10 appears to be an important
regulatory factor for NO production and presumably expression of iNOS
[15
]. The increased accumulation of macrophages in the
IL-10-/- mouse airway suggests that alveolar macrophages
may be an important source of this up-regulation of iNOS and NO
production and further confirms the critical role played by macrophages
in the pathogenesis of allergic inflammation. This set of observations
is consistent with the concept that IL-10 has multiple regulatory
mechanisms in the pathogenesis of allergic inflammation and that
further elucidation of these mechanisms may be of benefit in
understanding the regulation of airway inflammation in asthma.
Received December 18, 2000; revised June 11, 2001; accepted June 27, 2001.
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in human monocyte IL-10 synthesis J. Immunol. 151,6853-6861[Abstract] in murine macrophages Biochem. Biophys. Res. Commun. 182,1155-1159[Medline]1 J. Immunol. 165,4040-4050 and tumor necrosis factor- is enhanced by interleukin-10 Eur. J. Immunol. 23,2045-2048[Medline]This article has been cited by other articles:
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