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: Interactions in Lung Inflammation1
*
Department of Medicine, Harlem Hospital Center, Harlem Lung Center, Columbia University, New York, NY 10037;
DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304;
Genetics Institute, Cambridge, MA 02140;
Lung Biology Center and
¶ Howard Hughes Medical Institute, Departments of Medicine and
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Pathology, University of California, San Francisco, CA 94143;
#
Veterans’ Affairs Medical Center and Allergy Immunology Division, Medical College of Wisconsin, Milwaukee, WI 53295; and
**
Department of Pathology, Columbia University of Physicians and Surgeons and St. Luke’s-Roosevelt Hospital, New York, NY 10019
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IL-13, on the responses in the lungs. In a mouse model of airway
inflammation induced by mixed T cell responses, the number of Th1
(IFN--positive) cells was found to be negatively correlated with
airway hyperreactivity. In these mice, blockade of IL-13 partially
inhibited airway hyperreactivity and goblet cell hyperplasia but not
inflammation. In contrast, in mice that responded with a polarized Th2
response to the same Ag, blockade of IL-13 inhibited airway
hyperreactivity, goblet cell hyperplasia, and airway inflammation.
These results indicated that the presence of IFN- would modulate the
effects of IL-13 in the lungs. To test this hypothesis, wild-type mice
were given recombinant cytokines intranasally. IFN- inhibited
IL-13-induced goblet cell hyperplasia and airway eosinophilia. At the
same time, IFN- and IL-13 potentiated each other’s effects. In the
airways of mice given IL-13 and IFN-, levels of IL-6 were increased
as well as numbers of NK cells and of CD11c-positive cells expressing
MHC class II and high levels of CD86. In conclusion, IFN- has
double-sided effects (inhibiting some, potentiating others) on
IL-13-induced changes in the lungs. This may be the reason for the
ambiguous role of Th1 responses on Th2 response-induced lung
injury. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental models have demonstrated that different pathways can induce the asthma phenotype, eosinophilic inflammation, goblet cell hyperplasia, and airway hyperreactivity (reviewed in Refs. 3, 4, 5). However, these pathways are linked by the requirement for Th2 responses. Th2 responses can mediate inflammation, goblet cell hyperplasia, and airway hyperreactivity indirectly by producing cytokines such as IL-5, which is critical for lung eosinophilia (reviewed in Refs. 5, 6). Th2 cells can also mediate these signs directly by producing cytokines such as IL-4, IL-13 (7, 8, 9), or IL-9 (10).
However, in human subjects, chronic inflammatory diseases of the lungs
are associated with a more complex immune response that is, in many
cases, not polarized. Asthma, for example, presents with a complex
clinical and pathological phenotype (11) and with a
variable T cell response to allergens. Although Th2 cytokines and Th2
cells are present in the lungs of asthmatic patients (reviewed in Refs.
12, 13), simultaneous Th1 responses have been demonstrated
as well (reviewed in Ref. 14). In the airways of these
patients, numbers of Th1 cells, levels of IFN-, and activation of an
IFN--induced signaling molecule were detected
(15, 16, 17).
How Th1 and Th2 responses affect each other in the lungs is an unresolved question (reviewed in Refs. 18, 19). This question is important because originally it was believed that Th2 and Th1 responses would antagonize and thereby ameliorate each other’s pathological consequences. Following this view, the elicitation of a Th1 response to allergens, for example by vaccination, could be a therapeutic avenue (reviewed in Ref. 20). However, Th1 responses are thought to be critical instigators of some types of chronic lung inflammation, for example, hypersensitivity pneumonitis (21). Furthermore, Th1 responses, when present together with Th2 responses, have been shown to induce considerable lung inflammation (22, 23, 24, 25). In human subjects, this question is similarly unresolved. For example, successful allergen immunotherapy was associated with increased (26) or decreased Th1 responses (27). Furthermore, therapy with IL-12, a Th1-inducing cytokine, was not successful in reducing airway hyperreactivity or the late asthmatic response (28).
We have used different mouse models of allergen- and cytokine-induced
lung injury to address this problem. Our studies were focused on two
central mediators, IL-13, a critical Th2 mediator, and IFN-, a
critical Th1 mediator. We show that IFN- simultaneously inhibits
some, while potentiating other, effects of IL-13.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinase-activating gene (RAG)3 1-deficient (RAG1-/-) (C57BL/6 backcross) or RAG2-/- mice (129SvEv backcross) were purchased from The Jackson Laboratory (Bar Harbor, ME) or Taconic Farms (Germantown, NY), respectively. Wild-type mice were purchased from The Jackson Laboratory (C57BL/6) or from Taconic Farms (129SvEv). The mice were kept in barrier facilities at DNAX Research Institute (Palo Alto, CA), at the Gladstone Institute (University of California, San Francisco, CA), or in the Antenucci Building, St. Luke’s Roosevelt Hospital (New York, NY). By using monitoring with sentinel mice, the colonies were shown to be free of commonly tested mouse pathogens.
Isolation of donor T cells
Splenic CD4+ T cells were isolated as described (8, 29, 33). Briefly, single-cell suspensions were prepared by pressing tissues through 100-µm steel mesh filters followed by straining through 70-µm nylon filters. Red cell lysis was followed by magnetic depletion using lineage-specific mAbs (B220 (B cells), 8C5 (neutrophils), Mac-1 (monocytes and macrophages), Ter119 (erythrocytes), and anti-CD8 (CD8+ T cells)) and goat anti-rat IgG (Fc)- and anti-rat IgG (H and L)-coated magnetic beads (PerSeptive Diagnostics, Cambridge, MA). Remaining cells were labeled with CD4-FITC and Thy-1.2-PE (BD PharMingen, San Diego, CA) and sorted using instruments from Becton Dickinson (DNAX Research Institute and Columbia University, New York, NY) or a MoFlo multi-laser sorter high speed sorter, Cytomation (University of California). The purity of CD4+ T cells was typically >97%. In experiments that analyzed cells for the activation marker, CD62 L-selectin (CD62L), 82.44% ± 0.98% of the CD4+ T cells expressed CD62L at high levels, and 17.5% ± 0.97% expressed CD62L at low levels. For some experiments, positive magnetic bead selection was used (Miltenyi Biotech, Auburn, CA). Briefly, spleen cell suspensions were labeled with anti-CD4 mAb coupled to paramagnetic microbeads. Magnetically retained cells were >94% CD4+. Purified CD4+ T cells were injected i.p. at 2–5 x 106/mouse. The immunization protocol was started 3–6 days after T cell transfer.
Immunization
Asperigillus fumigatus Ag extract (Asp Ag) was prepared free of living organisms as described (30). Ag sensitization was conducted by priming with Asp Ag (100 µg in 200 µl PBS) given i.p. three times at 4-day intervals followed by intranasal (i.n.) challenge 4 days later (31). Challenges with Asp Ag (100 µg in 50 µl PBS) or control (PBS) were given to mice lightly anesthetized with isofluorane or methofane. Challenges were given i.n. two times (6 days apart) or three times (4 days apart).
Inhibition of IL-13
Inhibition of IL-13 was done as previously described
(8). Groups of T cell-reconstituted
RAG1-/- mice (C57BL/6 background) or of C57BL/6
wild-type mice were primed with Asp Ag i.p. on days -11, -7, and -4.
On days 0, 1, 4, 5, 6, 8, 9, and 10, the mice were given 0.325 mg of
IL-13R
2-Fc fusion protein (IL-13R-Fc) (32) or control
human Ig i.p. Challenges with Asp Ag i.n. were given on days 0 and 8.
On day 11, the mice were analyzed for a variety of parameters as
indicated in the results section.
Challenge with recombinant cytokines
Challenge with recombinant cytokines was done as previously
described (8). Groups of naive, wild-type C57BL/6 mice
were given in a 50-µl volume of PBS i.n. one of the following: 1)
control BSA (low in endotoxin; Sigma, St. Louis, MO), 2) murine rIL-13
(Genetics Institute) and BSA, 3) murine rIFN (R&D Systems,
Minneapolis, MN) and BSA, or 4) rIL-13 and rIFN-. IL-13 was used at
a dose of 5 µg/mouse at days 1, 3, and 5 as described (7, 8). IFN- was given at a dose of 1.25 µg/mouse mixed with
IL-13 and was additionally given (mixed with BSA) at days 0, 0.5, 2,
2.5, 4, and 4.5. The groups of mice that did not receive IFN- were
given BSA on days 0, 0.5, 2, 2.5, 4, and 4.5.
Airway hyperreactivity
Airway hyperreactivity was determined as described (8, 31, 33). Briefly, mice were anesthetized with etomidate (30 mg/kg), and the tracheas were cannulated. The mice were ventilated with 100% oxygen at physiologic tidal volumes (9 µl/g) and paralyzed using pancuronium bromide. A catheter was inserted into the tail vein, and the mice were placed inside of a plethysmograph. Increasing doses of acetylcholine (0.01–10 µg/g body weight) were administered i.v. until airway resistance reached at least 200% over baseline. From these data, the concentration of acetylcholine that produced a 200% increase in airway resistance over baseline (PC200) was calculated.
Bronchoalveolar lavage (BAL)
BAL was obtained as described previously (8, 31, 33). Samples were obtained by washing the lungs three times with 1 ml HBSS. Total cells were counted in undiluted samples. Cytospin preparations were stained with Wright and Giemsa solutions and a differential cell count performed on 300 cells or more. BAL supernatants were stored frozen (-70°C). Cytokine levels were determined in unconcentrated wash fluids using specific ELISAs. Flow cytometric analysis of BAL cells was performed using FITC-labeled anti-CD8, PE-labeled anti-CD4, CyChrome-labeled anti-CD3, allophycocyanin (APC)-labeled anti-Thy1.2, PE-labeled anti-NK1.1, FITC-labeled anti-CD11c, PE-labeled anti-CD86, and biotinylated anti-I-Ab mAbs. The biotinylated mAb was visualized using CyChrome-labeled streptavidin. All reagents were obtained from BD PharMingen. The cells were analyzed on a FACSCalibur using CellQuest software (BD Biosciences, San Jose, CA). Using forward and side scatter, gates were set for lymphocytes or for all other cells except lymphocytes.
Analysis of lung sections
Analysis of lung sections was performed as described previously (8). After BAL, the lungs were distended by injecting 1 ml formalin (4% formaldehyde solution in PBS), removed, and fixed in formalin. Parasagittal slices were paraffin-embedded and cut at 2–4 µm thickness. Sections were stained with H&E or with the periodic acid Schiff (PAS) methods. The sections were assigned a random code to blind the examiner to the identity of each specimen. The lungs were first evaluated for the general nature of the lesions. Scoring was then performed at a magnification of x250 by examining at least 50 consecutive fields.
Goblet cells
Medium-sized airways were assessed in sections stained with PAS. Each lung was assigned a unit by computing the mean of the numerical scores. The numerical scores for the abundance of PAS-positive goblet cells (8) in each airway were determined as follows: 0, <5% goblet cells; 1, 5–25%; 2, 25–50%; 3, 50–75%; 4, >75%.
Inflammation
Lung sections stained with H&E were assigned a unit value for peribronchiolar/perivascular inflammation or for alveolar inflammation by computing the means of the numerical scores.
Peribronchiolar and perivascular inflammation. The numerical scores for each view-field were determined as follows: 0, normal; 1, few cells; 2, a ring of inflammatory cells 1 cell layer deep; 3, a ring of inflammatory cells 2–4 cells deep; 4, a ring of inflammatory cells of >4 cells deep.
Alveolar inflammation. The numerical scores for each view-field were determined as follows: 0, normal; 1, alveolar walls normal, few macrophages in alveoli; 2, mild thickening of alveolar walls and increased alveolar macrophages and eosinophils; 3, marked thickening of alveolar walls and alveolar multinucleated giant cells and eosinophils in 30–50% of the field; 4, same as 3 but in >50% of the field; 5, complete consolidation.
Intracellular cytokine staining
Intracellular cytokine staining was performed as described
previously (31). The lung tissues were pressed through
100-µm steel mesh filters, and the cells were then strained through
70-µm and 40-µm cell strainers. Total cell numbers were determined
by manual counting using a Neubauer chamber. The cells were put into
culture with PMA/ionomycin for 2 h followed by addition of
brefeldin A for 2 h. DNase I (Sigma) was added during the last 5
min of culture. The cells were recovered by vigorous pipetting,
strained (70-µm cell strainer), and fixed with formaldehyde.
Intracellular labeling was performed using PE-labeled 11B11 mAb for
IL-4 (BD PharMingen), FITC-labeled AN18 mAb for IFN- (a generous
gift from A. O’Garra, DNAX Research Institute), and biotinylated
anti-IL-13 Ab (R&D Systems) followed by streptavidin-PE and in
cells permeabilized with saponin. Cell surface staining was performed
with CyChrome-labeled anti-CD3 and APC-labeled anti-Thy1.2 mAbs
(BD PharMingen). The cells were analyzed on a FACSCalibur (BD
Biosciences) using CellQuest software.
For each lung sample, the negative gates for the intracellular staining were set using cells exposed intracellularly to FITC- and PE-labeled isotype control mAbs (BD PharMingen) followed by surface labeling with anti-CD3 and anti-Thy1.2 mAbs. For the staining with 11B11 and with AN18, the background was very low, 0.5% fluorescing cells. The specificity of staining with the anti-IL-13 Ab was tested using soluble IL-13 given in excess. Using this method, we determined that the background for this Ab was at 1.5% fluorescing cells.
Determination of inhibitory activity of IL-13 in BAL samples
The B9 mouse cell line, in which growth is dependent on
different cytokines, including IL-13, which has been used to determine
IL-13 bioactivity or of IL-13-inhibitor activity (32), was
used to determine whether BAL samples would contain an inhibitor of
IL-13 function, e.g., soluble endogenous IL-13R2.
The cells were grown in RPMI 1640 (supplemented with glutamine and
pyruvate), 10% v/v FBS, penicillin-streptomycin, 20 nM 2-ME, and IL-6
(
100 ng/ml) as growth factor. To determine IL-13 responsiveness and
IL-13 inhibitor concentrations, the cells were washed and then placed
into culture at 1 x 104 cells per well into
flat-bottom 96-well culture plates. A dose-response curve of serial
dilutions of IL-13 (0.1–10 ng/ml) was determined. Cell proliferation
was determined after 3 days of culture using the fluorometric MTT
method (34). A concentration of IL-13 on the linear part
of the dose-response curve (2.5 ng/ml) was used for additional
experiments. A standard curve for inhibitor activity was constructed by
incubating the B9 cells with IL-13 (2.5 ng/ml) and 4-fold serial
dilutions of rIL-13R2-Fc (2.5 µg/ml to 0.002 ng/ml). Because some
of the BAL supernatants contained IL-6, the samples were tested in the
presence of a neutralizing anti-IL-6 Ab (2.5 µg/ml; BD
PharMingen). The inhibitor assay was performed by incubating B9 cells
with BAL samples (at serial 2-fold dilutions, final concentration 1/4
to 1/64), IL-13, and anti-IL-6. Positive control wells contained B9
cells and IL-13, negative control wells B9 cells and no growth factor.
Inhibitor activity was expressed as nanograms per milliliter
IL-13R2-Fc-equivalents.
Statistical analysis
Statistical analysis was performed as described (8, 31, 33). For airway hyperreactivity, differences between groups of animals were analyzed using the two tailed, unpaired Student’s t test on the logarithmic values of PC200. For all other measurements, differences between groups of animals were analyzed by the unpaired, two-tailed Wilcoxon U test. Multiple groups were analyzed by ANOVA followed by the unpaired Bonferroni, multiple comparison’s test. Analysis of correlation was performed using Spearman’s rank correlation test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As we had previously found that reconstitution of T- and B
cell-deficient RAG-/- mice with
CD4+ T cells is necessary and sufficient for the
induction of the signs of asthma by allergen (33), this
system was used further to study the mechanisms involved in the
process. RAG-/- mice of the 129SvEv or C57BL/6
strains were reconstituted with highly purified syngeneic
CD4+ T cells from naive wild-type mice and then
sensitized with Asp Ag (33). This Ag induces marked airway
hyperreactivity and a uniform Th2 response in wild-type mice (Ref.
31 and Fig. 1
). In contrast
to wild-type mice, the lung T cell response to Asp Ag in reconstituted
RAG-/- mice was nonuniform (Fig. 1A). Eighty percent of the mice developed a mixed lung T
cell response (simultaneous presence of IL-4+ and
IFN-+ T cells); 10% a Th2-like response
(IL-4+ T cells); and 10% a Th1-like response
(IFN-+ T cells). The mixed T cell responses in
the lungs of sensitized, T cell-reconstituted
RAG-/- mice were characterized by a pronounced
increase in the numbers of Th1 (IFN-+) cells
(10.65 ± 1.83 x 105 vs 0.4 ±
0.1 x 105 IFN-+ T
cells per lung (p < 0.0001) in T
cell-reconstituted RAG-/- vs wild-type mice).
In contrast, similar numbers of Th2 cells (IL-4+
or IL-5+) were present in Ag-challenged T
cell-reconstituted RAG-/- mice and in wild-type
mice (4.3 ± 0.6 x 105 vs 2.4 ±
0.4 x 105 IL-4+ T
cells per lung). The total numbers of T cells in the lungs (Thy1.2, CD3
double-positive) were similar in Ag-challenged T cell-reconstituted
RAG-/- mice and in wild-type mice (39.5 ±
5.0 x 105 vs 23.2 ± 2.3 x
105 cells per lung).
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Airway hyperreactivity was induced to a much more variable degree
in reconstituted RAG-/- mice relative to
wild-type mice (Fig. 1
B). There was a significant, but
nonlinear, negative correlation between the number of Th1 cells
(IFN-+) in the lungs and airway
hyperreactivity (Spearman’s rank correlation test, p
< 0.05; Fig. 1B). Airway hyperreactivity was induced to a
significant degree in those sensitized RAG-/-
mice that had <5 x 105
IFN-+ T cells in their lungs. Airway
hyperreactivity was also induced in some sensitized, reconstituted
RAG-/- mice that had >5 x
105 IFN-+ T cells in the
lungs; however, the group mean was not different from unsensitized
controls. Numbers of Th2 cells (IL-4+ or
IL-5+) in the lungs were not correlated with
airway hyperreactivity, or with numbers of Th1
(IFN-+) cells in the lungs (Fig. 1B).
Both strains of T cell-reconstituted RAG-/-
mice responded similarly. RAG1-/- mice (C57BL/6
background) or RAG2-/- mice (129SvEv
background) were reconstituted with CD4+ T cells
from syngeneic, naive, wild-type mice. Groups of 8–15 mice were used
for this comparison. Upon sensitization, both strains of mice developed
a robust Th2 response in the lungs (mean ± SEM: 3.5 ±
0.8 x 105 or 3.8 ± 0.5 x
105 IL-4+ T cells; 8.2
± 1.2 x 105 or 6.6 ± 1.1 x
105 IL-5+ T cells). Both
strains of mice showed a variable Th1 response in the lungs (mean,
range: 5.5, 0.1–23.1 x 105 or 5.9,
0.3–24.5 x 105
IFN-+ T cells). Airway hyperreactivity was
induced to a similar variable degree (mean, range of
PC200: 0.89, 0.2–2.0 or 0.57, 0.1–2 µg/g).
The variability in the immune response to Asp Ag seen in both strains
of T cell-reconstituted RAG-/- mice is most
probably due to the innate immune system (immaturity of dendritic cells
and presence of a large population of NK cells relative to numbers of T
cells (our unpublished data)).
This model was then used to examine which molecules are critical for the airway physiologic and inflammatory changes in allergen-induced lung disease characterized by mixed T cell responses. We hypothesized that IL-13 would be the critical effector molecule as shown in Th2-induced asthma models (7, 8).
IL-13: a partial mediator of allergen-induced lung injury in T cell-reconstituted RAG-/- mice
Groups of T cell-reconstituted RAG1-/- and wild-type mice were primed i.p. with Asp Ag followed by challenge i.n. An IL-13 inhibitor (IL-13R-Fc) (32) or control protein was given during the i.n. challenge period.
Airway hyperreactivity (Fig. 2A).
IL-13R-Fc potently inhibited airway hyperreactivity in wild-type mice.
In T cell-reconstituted RAG-/- mice, Asp Ag
induced airway hyperreactivity in some mice. Because of the variability
of airway hyperreactivity, and the relative small number of animals
examined, the group means did not reach statistical significance.
T cell-reconstituted RAG-/-mice that received
IL-13R-Fc had a trend to reduced airway hyperreactivity that was
statistically not significant.
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B).
IL-13R-Fc inhibited Ag-induced goblet cell hyperplasia in wild-type
mice potently. Similarly, IL-13R-Fc significantly inhibited goblet cell
hyperplasia in Ag-sensitized, T cell-reconstituted
RAG-/- mice. However, the relative inhibition
of goblet cell hyperplasia by IL-13R-Fc was smaller in reconstituted
RAG-/- mice as compared with wild-type
mice.
BAL eosinophilia (Fig. 2C).
IL-13R-Fc significantly inhibited Ag-induced BAL-eosinophilia in
wild-type mice. In contrast, IL-13R-Fc did not reduce BAL eosinophilia
in T cell-reconstituted RAG-/- mice.
BAL neutrophilia (Fig. 2D).
In wild-type mice, neutrophils were an appreciable constituent of BAL
fluid on the first day after giving Ag (data not shown), but
neutrophilia was mostly resolved 4 days after Ag challenge (Fig. 2D). In T cell-reconstituted RAG-/-
mice, Ag induced prolonged BAL neutrophilia (Fig. 2D).
Injections of IL-13R-Fc did not affect BAL neutrophilia.
Inflammation (Figs. 2, E and F, and
3, A–F).
Control wild-type mice did not show signs of inflammation. Control T
cell-reconstituted RAG-/- mice demonstrated
mild inflammation of the lungs. This inflammation could reflect an
increased state of activation of the transferred T cells as has
been observed in T cell transfer models used to study inflammatory
bowel disease (35). Ag sensitization induced
peribronchiolar, perivascular, and alveolar inflammation of similar
type and extent in wild-type and in T cell-reconstituted
RAG-/- mice. Peribronchiolar and perivascular
inflammation was characterized by infiltrates with mononuclear cells
and eosinophils. Alveolar lesions were patchy and varied in severity.
Mild lesions were characterized by filling of the alveoli with
increased numbers of inflammatory cells, most of which were mononuclear
cells. Severe lesions included thickening of alveolar walls by
eosinophils and occasional lymphoid cells. The type II cells showed
hyperplasia, and alveoli were filled with a mixture of eosinophils,
macrophages, and multinucleated giant cells.
Alveolar inflammation (Figs. 2E and 3,
C–F).
In wild-type mice, IL-13R-Fc potently reduced alveolar inflammation. In
contrast, IL-13R-Fc did not affect alveolar inflammation in T
cell-reconstituted RAG-/- mice.
Peribronchiolar and perivascular inflammation (Figs. 2F and 3, C–F).
IL-13R-Fc did not affect peribronchial and perivascular inflammation in
wild-type or T cell-reconstituted RAG-/-
mice.
Correlation of scores of lung inflammation and airway hyperreactivity in individual T cell-reconstituted RAG-1-mice
To control for the possibility that in this series of experiments, low airway hyperreactivity would be the result of an insufficient immune response, the data were analyzed for correlation with lung inflammation. There was no correlation between airway hyperreactivity and peribronchial inflammation (p = 0.728); or alveolar inflammation (p = 0.148); or goblet cell hyperplasia (p = 0.755). For example, the mice with the least inflammation (alveolar inflammation scores of 0.857 and 1.472; peribronchial inflammation scores of 0.667 and 1.382) had PC200 values of 0.222 µg/g (the most hyperreactive) or 0.514 µg/g (the fourth most hyperreactive). The two mice with the most inflammation (alveolar inflammation scores of 2.4 and 2.5; peribronchial inflammation scores of 2.4 and 2.65) had PC200 values of 0.992 µg/g (not hyperreactive) or 0.381 µg/g (the third most hyperreactive mouse).
IL-13+ T cells in the lungs, and IL-13-inhibitor activity in BAL supernatants
Wild-type and T cell-reconstituted RAG-/-
mice were examined for the presence of IL-13+ T
cells and IL-13-inhibitor activity in the lungs to determine whether
the diminished effects of IL-13 inhibition in T cell-reconstituted
RAG-/- mice was due to a lack of an
IL-13-response to Ag or due to insufficient transfer of the IL-13R-Fc
to the lungs. Similar numbers of IL-13+ T cells
could be detected in the lungs of sensitized wild-type or reconstituted
RAG-/- mice (mean, range: 5.485, 1.1–11.1
x 105 or 12.2, 0.44–27.7 x
105 IL-13+ T cells,
respectively). At the time point of analysis (4 days after the last
sensitization), IL-13, like IL-4, IL-5, or IFN-, could not be
detected by ELISA. A cytokine-dependent cell line, B9, was used to
detect IL-13-inhibitor activity in BAL supernatants. Approximately 60%
of the samples from control or Ag-sensitized wild-type and T
cell-reconstituted RAG-/- mice contained
activity that inhibited IL-13-induced growth of B9 cells. However, that
inhibitory activity was at the detection limit of the assay. In
contrast, all samples from mice that were given IL-13R-Fc inhibited
IL-13-induced growth of B9 cells. All of the samples had high
inhibitory activity that was detectable up to the highest dilution of
the BAL samples examined. The IL-13R-Fc inhibitor equivalent activity
was 8.4 (mean), 2.6–12.4 µg/ml (range) in wild-type mice and 12.4
µg/ml in T cell-reconstituted RAG-/- mice.
However, the exact amount of IL-13-inhibitory activity could not be
determined in the T cell-reconstituted RAG-/-
mice because 7 of 10 BAL samples contained IL-6 by ELISA (group
mean ± SEM, 0.56 ± 0.22 ng/ml). In contrast, IL-6 could not
be detected in any of the BAL samples from wild-type mice given
IL-13R-Fc. Because IL-6 is another growth factor for B9 cells, most BAL
samples from reconstituted RAG-/- mice promoted
growth of B9 cells, even in the presence of the anti-IL-6 Ab.
However, at the highest BAL dilution tested, all samples from
RAG-/- mice given IL-13R-Fc inhibited the
growth of B9 cells completely.
These data show that the relative diminished potency of IL-13R-Fc to
inhibit pathology in T cell-reconstituted
RAG-/- mice relative to wild-type mice was not
due to a lack of IL-13-expressing T cells in the lungs or to an
insufficient availability of the IL-13 inhibitor in the lungs.
Therefore, we hypothesized that a molecule made by Th1 cells during the
course of the mixed T cell response in the lungs would modulate the
function of IL-13. We hypothesized that this molecule was IFN-
because IFN- has been shown to inhibit allergen-induced lung injury
(36, 37, 38, 39, 40, 41, 42). To test this hypothesis, wild-type mice were
given recombinant cytokines.
IFN- inhibits and at the same time potentiates effects of IL-13
in the lungs
Wild-type mice were given recmbinant IFN- and IL-13, or each
cytokine alone, or BSA i.n. The mice were analyzed 1 day after the last
cytokine challenge. BAL fluids were analyzed for IL-13 and IFN- to
ensure that differences between groups of mice were not due to
insufficient exposure to cytokines. Both IL-13 and IFN- levels were
similar in groups of mice that were challenged with either IL-13,
IFN-, or with IL-13 and IFN- (mean ± SEM: 17.3 ± 1.2
vs 17.7 ± 2.6 ng/ml IL-13; and 14.8 ± 3.9 vs 10.8 ±
4.8 ng/ml IFN- comparing mice challenged with respective single
cytokines vs both cytokines together). Differences between groups of
mice were also not due to a large induction of TNF- or IL-12
production, because these cytokines could not be detected in BAL
samples by ELISA (data not shown).
Inhibitory effects
IFN- inhibited goblet cell hyperplasia (Fig. 4A), BAL eosinophils (Fig. 4B), and BAL neutrophils (Fig. 4B) induced by
IL-13. There was a trend toward a decrease in the numbers of
BAL- CD4+ T cells in mice
challenged with IL-13 and IFN- relative to mice challenged with
IL-13 (mean ± SEM: 0.66 ± 0.13 x
104, or 1.03 ± 0.20 x
104 CD4+ T cells/BAL,
respectively).
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There was a trend to increased peribronchiolar and alveolar
inflammation in mice given IL-13 and IFN- relative to mice
challenged with IL-13 (Figs. 4C and
5, A–D). Both parameters of
inflammation were significantly increased relative to mice given BSA or
mice challenged with IFN-. Numbers of NK cells in the BAL were
significantly increased in mice given IL-13 and IFN- relative to all
other groups of mice (Fig. 4B). Levels of IL-6 were
increased in mice given IL-13 and IFN- relative to all other groups
of mice (Fig. 4D). Numbers of cells expressing high levels
of Ag-presentation molecules were increased in mice given IL-13 and
IFN- relative to all other groups of mice (Fig. 6). These cells were detected using
anti-CD11c (a molecule expressed by dendritic cells), MHC class II,
and CD86 (B7-2) Abs. These Abs marked cell subpopulations that were
distinct for each group of mice (Fig. 6A). The percentage of
cells that coexpressed MHC class II, CD11c, and high levels of CD86 was
significant higher in mice challenged with IL-13 and IFN- relative
to mice challenged with IL-13 (Fig. 6Ah; p
< 0.001). The numbers of these cells expressing MHC class II,
CD11c, and high levels of CD86 were significantly increased in the BAL
of mice challenged with IL-13 and IFN- as compared with all other
groups of mice (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ T cells in the lungs. The
correlation was such that airway hyperreactivity was induced on a group
level in mice that had <5 x 105
IFN-+ T cells in the lungs. These data confirm
reports of many other groups that the presence of large amounts of
IFN- (by administering IFN-, IL-12, or IL-18) in the lungs
inhibits the development of airway hyperreactivity induced by Th2
responses in the lungs (38, 39, 41, 43, 44, 45, 46, 47, 48). Our data show that IL-13 was only a partial mediator of lung injury induced by mixed T cell responses. This is in contrast to Th2-mediated asthma models due to OVA sensitization, in which IL-13 has been shown to play a central role (7, 8, 49). Our results confirm this conclusion for Th2 models using a different Ag. In mice that developed polarized Th2 responses to Asp Ag, airway hyperreactivity, goblet cell hyperplasia, and inflammation were all blocked by IL-13R-Fc. However, in mice that developed mixed T cell responses (Th1 and Th2) to Asp Ag, blockade of IL-13 reduced the numbers of mice that developed airway hyperreactivity, reduced goblet cell hyperplasia, but did not affect inflammation. Furthermore, in the mice with lung injury due to mixed T cell responses, the extent of inhibition of airway hyperreactivity and goblet cell hyperplasia was relatively smaller than that seen in mice that had Th2-induced lung injury. This may be because airway hyperreactivity developed in a smaller percentage of mice, and because goblet cell hyperplasia developed to a smaller degree in mice with mixed T cell responses. Alternately, the decrease in relative inhibition may be due to mediators other than IL-13, which contributed to airway hyperreactivity and goblet hyperplasia in these mice.
IL-13 inhibition clearly reduced airway and parenchymal inflammation in
lung injury due to Th2 responses but not in lung injury due to mixed T
cell responses (Fig. 2). This confirms published results showing
IL-13’s role in airway inflammation (7, 8), in
parenchymal inflammation (9), and in emphysema
(50). Therefore, the most likely explanation for the
inability to demonstrate a role for IL-13 in inflammation in the
model of mixed T cell responses was that a molecule made by Th1 cells
during the course of the mixed T cell response in the lungs would
modulate the function of IL-13. We hypothesized that this molecule was
IFN-.
Using recombinant cytokines, our data demonstrate that IFN- has
double-sided effects on IL-13-induced lung injury. IFN- inhibited
IL-13-induced goblet cell hyperplasia and the infiltration of the
airways with eosinophils and neutrophils. This result provides a
mechanistic explanation for the down-regulation of the signs of asthma
in allergen-induced lung disease by IFN- that has been described
(36, 37, 38, 39, 41, 42, 46, 47). The inhibition of IL-13
functions in the lungs by IFN- is analogous to the inhibition of
IL-13-mediated worm expulsion by IFN- in the intestines
(51). The inhibition by IFN- of IL-13 functions
in the lungs may recapitulate on the level of a whole organ, the
IFN--mediated inhibition of IL-13-induced IL-1R-antagonist
expression in macrophages (52), or the IL-13-induced IgE
production in human B cells (53).
In contrast, IFN- potentiated the effects of IL-13-induced lung
injury. Mice challenged with IL-13 and IFN- had increased numbers of
cells expressing high levels of Ag-presentation molecules, NK cells,
and increased levels of IL-6 in the BAL. Lung inflammation
(peribronchiolar and alveolar scores) showed an increased trend
relative to mice challenged with IL-13 and was significantly increased
relative to mice challenged with IFN-. Although we were surprised
that mice challenged with IFN- did not have higher numbers of cells
expressing high levels of Ag-presenting molecules in the BAL, this
result is supported by a report by Suda et al. (54),
showing that IFN- induces increased expression of MHC class II in
resident lung dendritic cell precursors but not increased migration of
circulating dendritic cells to the lungs. The high levels of IL-6
present in BAL samples from mice challenged with IL-13 and IFN- may
be the result of a synergistic effect of IFN- and IL-13 on IL-6
production in bronchial epithelial cells (55). In these
mice, IL-6 is most probably not made by macrophages as IL-13 inhibits
IFN--induced IL-6 production in macrophages (56). The
presence of high numbers of NK cells in the samples of mice challenged
with IL-13 and IFN- is most probably due to a very specific
regulation of chemokines and chemokine receptors that promotes
migration of some cells and inhibits migration of others.
The additive and synergistic effects of IL-13 and IFN- in the lungs
may regulate subsequent immune and inflammatory response to inhaled
Ags. We do not know if the high levels of IL-6 present in the airways
of mice exposed to IL-13 and IFN- have a pro- or
anti-inflammatory role. Although IL-6 has been described to be
important for the development of Th2 responses (57), it
has also been described to inhibit allergen-induced airway disease
(58). The same is true for NK cells, as they can
differentiate to secrete distinct patterns of cytokines
(59). Although NK cells have been shown to be necessary
for the full elicitation of lung injury in some allergen-induced models
(60, 61), these cells are also important for protection
from aberrant chronic inflammation (62). The increase in
the numbers of cells expressing high levels of Ag-presenting molecules,
MHC class II, and CD96 may predispose the lungs for increased responses
to inhaled Ags because CD86 is a critical costimulatory molecule,
particularly for the induction of Th2 responses (63, 64, 65, 66).
Increased capability of Ag presentation in airways exposed to IL-13 and
IFN- could explain why inflammation is more intense in Ag-challenged
mice that were given Ag-specific Th2 and Th1 cells by cell transfer
(22, 23, 24) or why IFN- is requisite for allergen-induced
inflammation in some mouse models (67, 68).
In conclusion, although IL-13 is the major effector cytokine of goblet
cell hyperplasia, airway hyperreactivity, and inflammation in lung
injury induced by polarized Th2 responses, IL-13 is only a partial
mediator of the changes in the lungs induced by mixed T cell responses.
This divergence is most probably due to the presence of the inhibitor
and potentiator of IL-13-mediated changes in the lungs, IFN-.
Whereas IFN- limits IL-13-induced goblet cell hyperplasia and
eosinophilic inflammation, it increases numbers of cells expressing
high levels of Ag-presenting molecules, NK cells, and IL-6 in the
airways. By this mechanism, IFN- and IL-13 induce a distinct type of
inflammation and may prepare the lungs for increased responses to
inhaled Ags.
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Acknowledgments |
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2-Fc fusion protein. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Gabriele Grünig, St. Luke’s-Roosevelt Hospital, 432 West 58th Street, Laboratory 504, New York, NY 10019. E-mail address: gg398{at}columbia.edu
3 Abbreviations used in this paper: RAG, recombinase-activating gene; APC, allophycocyanin; Asp Ag, Aspergillus fumigatus Ag; BAL, bronchoalveolar lavage; i.n., intranasal; IL-13R-Fc, IL-13 receptor 2-Fc fusion protein; PAS, periodic acid Schiff; PC200, provocative dose 200, the dose of acetylcholine that induces a 200% increase over baseline resistance; CD62L, CD62 L-selectin.
Received for publication December 5, 2000. Accepted for publication May 21, 2001.
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