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*
Section of Pulmonary and Critical Care Medicine, Department of Internal Medicine, and
Department of Pathology, Yale University School of Medicine, New Haven, CT 06520; and Pathology and Laboratory Medicine Service, Veterans Administration-Connecticut Health Care System, West Haven, CT 06516
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and -2,
and mRNA, increased bronchoalveolar lavage (BAL) TGF-ß1,
and exaggerated airway responses to aerosolized methacholine. In
contrast, CC10-IL-6 mice, on both C57BL/6 and BALB/c backgrounds,
manifest diminished inflammation and eosinophilia, decreased levels of
IL-4, IL-5, and IL-13 protein and mRNA, and decreased levels of
bronchoalveolar lavage TGF-ß1. IL-6 also decreased the
expression of endothelial VCAM-1 and airway responsiveness to
methacholine in these animals. These alterations in the
IL-6-/- and CC10-IL-6 mice were not associated with
significant decreases or increases in the levels of IFN-
,
respectively. These studies demonstrate that endogenous and exogenous
IL-6 inhibit aeroallergen-induced Th2 inflammation and that this
inhibition is not mediated by regulatory effects of IFN-. IL-6 may
be an important anti-inflammatory, counterregulatory, and healing
cytokine in the airway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-6 is a pleiotropic cytokine that is produced during the course of a wide variety of infectious, inflammatory, and traumatic disorders (8, 9, 10, 11, 12, 13). Exaggerated production of IL-6 is well documented in asthma and asthmatic tissues (10, 14, 15, 16). However, the role that IL-6 plays in asthma is not clear because equally cogent arguments can be made for pro- and anti-inflammatory effects of this important cytokine moiety. The pro-inflammatory effects include the essential role of IL-6 in IgA production (17), turpentine-induced tissue inflammation (18), Lyme arthritis (19), tumor infiltration (20), Ag-induced arthritis (21), carrageenan-induced pleural inflammation (11), and the control and/or irradication of infections caused by Mycobacterium tuberculosis vaccinia virus, Listeria, vesicular stomatitis virus, and Candida (13, 22, 23). In contrast, anti-inflammatory effects of IL-6 have been demonstrated in systemic and pulmonary models of endotoxin-induced injury (24, 25), immune complex lung injury (12), pneumococcal pneumonia (26), and staphylococcal enterotoxin-induced toxic shock (27). In addition, IL-6-induced pro- and anti-inflammatory effects can be tissue and stimulus specific (18, 28, 29), and IL-6 can augment (19, 30) or inhibit (13, 21, 22) Th2 tissue inflammation. Despite the well-documented dysregulation of IL-6 in asthma and the importance of chronic Th2 inflammation in asthma, the ability of IL-6 to regulate Th2 inflammation in the airway has not been adequately investigated.
To characterize the anti-inflammatory mechanisms involved in airway healing and repair and the role(s) of IL-6 in these mechanisms, we characterized the responses induced by aeroallergen in wild-type (WT)3 mice, mice that lack the ability to make IL-6 (IL-6-/- mice) and mice in which IL-6 is overexpressed in an airway/lung-specific fashion (CC10-IL-6 mice). These studies demonstrate that IL-6 is a potent multifaceted inhibitor of aeroallergen-induced responses because tissue and bronchoalveolar lavage (BAL) inflammation, eosinophilia, and Th2 cytokine production are exaggerated in the IL-6-/- mice and inhibited the CC10-IL-6 animals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice that were unable to make IL-6
(IL-6-/-) and that overexpressed IL-6 in the
lung/airway under the influence of the CC10 promoter (CC10-IL-6) were
used in these studies. The IL-6 knockout mice were obtained on a
C57BL/6 background with appropriate C57BL/6 WT controls from The
Jackson Laboratory (Bar Harbor, ME). Before use their (-/-) genotype
was confirmed via PCR analysis. The CC10-IL-6 mice that were used were
generated in our laboratory (31). The methods that were
used to generate these mice, the organ specificity of transgene
expression, and the alterations induced by IL-6 have been described
previously (31, 32). These mice were initially generated
using CBA x C57BL/6 animals. They were subsequently bred for 
8
generations onto C57BL/6 or BALB/c backgrounds and C57BL/6 or BALB/c
transgene (-) littermates, as appropriate, were used as WT
controls.
OVA sensitization and challenge
OVA sensitization and challenge were accomplished using modifications of the protocols previously described by Yang et al. (33). In brief, 6- to 8-wk-old WT mice, CC-10-IL-6 transgene (+) mice, and/or IL-6-/- mice received i.p. injections containing 20 µg of turkey OVA (Sigma, St. Louis, MO) complexed to alum (Resorptar; Indergen, New York, NY). This process was repeated 5 days later. After an additional 7 days, some animals were sacrificed (time = 0) and others received aerosol challenge with OVA (1% w/v) in endotoxin-free PBS or endotoxin-free PBS alone. This was accomplished in a closed 27 x 20 x 10 cm plastic aerosol chamber in which the mouse was placed for 40 min. The aerosol was generated via an Omron NE-U07 ultrasonic nebulizer (Omron Healthcare, Vernon Hills, IL). Mice were sacrificed 4, 24, 48, 72 h, or 7 days after aerosol exposure.
Bronchoalveolar lavage
BAL was performed as previously described (34). In brief, after anesthesia, a median sternotomy was performed, the trachea was dissected free from the underlying soft tissues, and a 0.6-mm tube was inserted through a small incision in the trachea. BAL was performed by perfusing the lungs in situ with 0.6 ml PBS and gently aspirating the fluid back. This was repeated three times. The samples were then pooled and centrifuged, and cell numbers and differentials were assessed. The cell-free BAL fluid was stored at -70°C until used.
Histologic analysis
Mice were anesthetized, a median sternotomy was performed, the trachea was dissected free and cannulated as described above, and the pulmonary vascular tree was perfused with calcium- and magnesium-free PBS (pH 7.40) via a catheter in the right heart. The lungs were then inflated to a 25-cm pressure with 10% formalin in PBS (pH 7.40), removed from the chest, postfixed in 10% formalin in PBS for 24 h, placed in paraffin, and sectioned. Hematoxylin and eosin and Congo red stains were performed in the Department of Pathology of Yale University School of Medicine.
Quantification of tissue inflammation
Six to eight images of each lung section were captured at x25 final magnification on an Olympus (Tokyo, Japan) BH-2 microscope using a Sony DXC-760 MD camera attached to a Macintosh PPC8100/80 with a RasterOps 24 MxTv frame grabber board. Images were collected at 16-bit color depth, 640 x 480 pixels, final magnification 0.75 pixels per micron using the software that came with the frame grabber board. They were then analyzed in NIH Image 1.62 using a computer-generated 18 line x 13 line overlay grid. Total intersections overlying inflamed lung parenchyma, excluding vessels and airways, were counted and compared with total intersections overlying parenchyma. The percentage of total points overlying inflamed lung per animal was then determined, and an average was made over each group.
Quantification of tissue eosinophils
Tissue eosinophilia was quantitated as previously described by our laboratory (35). In brief, on each Congo red stained slide, lungs were divided into six to eight rectangular areas using a 4-mm grid. From each area, the best-defined nontangentially cut bronchovascular area was selected at x40 final magnification using an Olympus BH-2 microscope. Eosinophils are not well visualized at that power. Eosinophils were counted at x200 final magnification. Area was quantitated using a rectangular 10-mm square reticule grid (American Optical, Buffalo, NY) inserted into one eyepiece. The number of lower left corner grid intersections overlapping the bronchovascular bundle was used as an approximation of area. The ratio of the total number of eosinophils to total area for each mouse lung was calculated, and the mean of these results was calculated for each experimental group. In our hands this method correlates well with eosinophils quantitated using tissue digestion.
Cytokine mRNA quantification: RNase protection assays (RPAs)
The levels of mRNA encoding Th1 and Th2 cytokines and chemokines were quantitated using RPA. Briefly, mice were sensitized and challenged, and their lungs were removed as described above. They were then digested in TRI-zol reagent (Life Technologies, Gaithersburg, MD), and total RNA was obtained by processing the tissues according to the manufacturer’s specifications. The levels of specific mRNA transcripts were then evaluated by RPA using Riboquant kits (PharMingen, San Diego, CA) according to the instructions provided by the manufacturer.
Cytokine quantitation
The levels of IL-4, IL-5, IL-13, IFN-, eotaxin, RANTES,
macrophage inflammatory protein (MIP)-1 MIP-2, JE/monocyte
chemottractant protein (MCP)-1, and TGF-ß1
protein were quantitated by ELISA using commercial kits according to
the instructions provided by the manufacturers (R & D, Minneapolis, MN;
Endogen, Cambridge, MA).
VCAM-1 immunohistochemistry (IHC)
Immunoreactive VCAM-1 was assessed by IHC as described by Yang et al. (33). Mice were sensitized, challenged with OVA, and sacrificed, then their lungs were perfused with 0.001 M periodate/0.075 M lysine/1% paraformaldehyde (PLP) and fixed overnight at 4°C followed by cryoprotection by consecutive 20-min incubations in graded cold sucrose solutions and inflated with 1 ml 40% OCT diluted in PBS. They were then embedded in 100% OCT in a cryomold and stored at -70°C until sectioning. Tissue sections were soaked in wash buffer (0.1 M phosphate buffer plus 0.01% Triton X-100) for 10 min, blocked in 3% BSA in wash buffer, and incubated with a 1:50 dilution of the primary Ab (biotinylated anti-mouse VCAM-1; PharMingen) or isotype control Ab for 1.5 h at 25°C. The slides were then washed, incubated with streptavidin-alkaline phosphatase (Zymed, San Francisco, CA) for 30 min, rewashed, developed with Fast Red, and counterstained with hematoxylin.
Physiological assessment
The baseline resistance and AHR in unrestrained, conscious animals was assessed by barometric plethysmography using whole-body plethysmography (Buxco Electronics, Troy, NY) as described by our laboratory (36) and others (37). In brief, mice were placed into whole-body plethysmographs and interfaced with computers using differential pressure transducers. Measurements were made of respiratory rate, tidal volume, and enhanced pause (Penh). Airways resistance is expressed as Penh = ((te/0.3 tr) - 1) x (2 Pef/3 Pif),where Penh = enhanced pause, te = expiratory time (in seconds), tr = relaxation time (in seconds), Pef = peak expiratory flow (in milliliters), and Pif = peak inspiratory flow (in milliliters per second). Increasing doses of methacholine were administered by nebulization for 120 s, and Penh was calculated over the subsequent 5 min. The Penh values for each mouse are reported as the percent increase over the Penh value obtained before the administration of any methacholine.
Statistical Analysis
Data are expressed as means ± SEM unless otherwise indicated. Data were assessed for significance using the Student’s t test or ANOVA as appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether endogenous IL-6 regulates
aeroallergen-induced responses in the lung, we compared the effects of
OVA in sensitized WT and IL-6-/- mice before
and at intervals after aerosol challenge. Aeroallergen sensitization
and challenge of WT mice caused an impressive increase in BAL
cellularity (Fig. 1
A). This
increase was noted within 4 h of challenge and was maximal 48
h after Ag exposure. Before aeroallergen challenge, macrophages were
the dominant cell in the BAL fluids from WT mice. However, Ag challenge
caused an impressive increase in BAL eosinophils. Enhanced eosinophil
recovery was noted within 4 h, peaked after 48–72 h, and was
still present 7 days after Ag exposure (Fig. 1B). Within
48 h of Ag challenge, eosinophils were the predominant
inflammatory cell in BAL fluid (Fig. 1C). This impressive
increase in BAL eosinophils was associated with a significant decrease
in the percentage of BAL cells that were macrophages (Fig. 1D). Comparable degrees of BAL inflammation were not seen in
WT animals that were not sensitized before aeroallergen challenge or
that received systemic sensitization and nebulized saline (data not
shown).
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A). BAL eosinophil recovery was also enhanced in the
IL-6-/- vs the WT mice at all time points after
Ag challenge. Twenty four hours, 48 h, and 7 days after Ag
challenge, BAL from IL-6-/- mice contained
6.7-, 2.8-, and 6.7-fold, respectively, greater numbers of eosinophils
than BAL from WT animals (p < 0.01 for all
time points) (Fig. 1B). As expected from this eosinophil
influx, the percentage of BAL cells that were eosinophils was
significantly greater and the percentage of BAL cells that were
macrophages was significantly lower in the
IL-6-/- than in the WT mice 4 and 24 h
after OVA exposure (Fig. 1, C and D). In
contrast, statistically significant differences in the percentage of
BAL cells that were granulocytes or lymphocytes were not noted in
comparisons of the WT and IL-6-/- mice at all
time points (data not shown).
OVA challenge of WT mice caused a peribronchiolar and perivascular
inflammatory response that contained significant tissue eosinophilia
(Fig. 2A). This response was
readily appreciated 24–48 h after OVA aerosol challenge and was not
seen in animals that were not sensitized before aeroallergen challenge
or that received systemic sensitization and nebulized saline (data not
shown). In accord with the BAL finding, the tissue response was
markedly enhanced in the IL-6-/- animals (Fig. 2). Compared with control WT mice, the IL-6-/-
mice had denser pulmonary infiltrates around vessels and in the
parenchyma. These infiltrates contained eosinophils and lymphocytes,
the latter of which appeared more activated than in WT mice with more
open chromatin and nucleoli. At 48 h after Ag challenge, the
inflammatory infiltrate in the tissues was 
2.5 times greater in
IL-6-/- mice than in WT controls. When the BAL
and tissue findings are viewed in combination, they demonstrate that
tissue and BAL inflammation and eosinophilia are markedly enhanced in
IL-6-/- as vs WT mice.
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Studies were next undertaken to determine whether endogenous IL-6
regulated OVA-induced Th2 cytokine production. IL-4, IL-5, and IL-13
were not appreciated in significant quantities in the BAL fluid of
sensitized unchallenged mice, sensitized mice challenged with saline,
or mice that were not sensitized and challenged with aerosol OVA (Fig. 3 and data not shown). IL-4, IL-5, and
IL-13 proteins were readily appreciated in BAL fluids, and the mRNA
moieties encoding these cytokines were readily appreciated in whole
lung RNA from sensitized WT mice 24 h after OVA exposure (Figs. 3 and 4). Significant levels of IL-4, IL-5,
and IL-13 protein and mRNA were not appreciated in sensitized
IL-6-/- mice in the absence of aerosol
exposure. However, aerosol exposure of sensitized IL-6-deficient mice
resulted in exaggerated Th2 responses. When compared with WT controls,
significantly increased levels of IL-4, IL-5, and IL-13 protein were
detected in the BAL fluids from IL-6-/- mice.
These differences were most prominent 24–48 h after Ag challenge
(p < 0.01 for all cytokines). At 24 h,
IL-6-/- mice produced 6.2-, 6.1-, and 14.1-fold
more IL-4, IL-5, and IL-13, respectively, than identically challenged
WT controls (Fig. 3). Similar increases in the levels of IL-4, IL-5,
and IL-13 mRNA were noted in lung RNA from
IL-6-/- vs WT mice (Fig. 4). These studies
demonstrate that the Th2 cytokines IL-4, IL-5, and IL-13 are produced
in an exaggerated fashion in OVA-sensitized and -challenged
IL-6-/- mice.
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Because IFN- can regulate Th2 cytokine production and effector
function (5, 38), studies were undertaken to quantitate
the levels of IFN- in lungs from sensitized and challenged
IL-6-/- and WT animals. The levels of IFN-
in the BAL fluids from WT mice before and after aeroallergen challenge
were at or below the limits of detection of our assay. Similarly, low
levels of IFN- were noted in the BAL fluids from the
IL-6-/- mice before OVA challenge (Fig. 5). Four and 24 h after Ag
challenge, a modest increase in the levels of BAL IFN- were noted in
the IL-6-/- vs the WT mice (Fig. 5A). However, these increases did not reach statistical
significance. Overall, the levels of IFN- produced by OVA-challenged
IL-6-/- mice were greater than or equal to the
levels in WT animals (data not shown). Thus, the exaggerated Th2
response in these mice is not due to decreased IFN- production.
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B). Similar levels of TGF-ß1 were
appreciated in IL-6-/- mice before Ag
challenge. In contrast, significantly increased levels of
TGF-ß1 were seen 4 h after OVA exposure.
At this time point, BAL fluid from IL-6-/- mice
contained 3.5-fold more TGF-ß1 than BAL from WT
mice (p < 0.05). Thus, the exaggerated Th2
response in IL-6-/- mice is associated with the
enhanced accumulation of TGF-ß1. Chemokine production in IL-6-/- and WT mice
We next assayed the levels of protein and mRNA for a variety of
chemokines that play important roles in this biologic system. In
sensitized WT mice, the basal levels of BAL fluid eotaxin, MIP-1,
MIP-2, and JE/MCP-1 were at or near the limits of detection of our
ELISA, and OVA challenge either did not alter or modestly increased the
levels of these cytokine moieties (Fig. 6). In IL-6-/-
mice, exaggerated levels of these chemokines were noted after OVA
challenge. This effect was most prominent 4 and 24 h after OVA
challenge (p < 0.01 for all cytokines at these
time points). At the time points of maximal chemokine production by WT
mice, BAL fluids from IL-6-/- mice contained
6.0-, 4.1-, 7.1-, and 35.2-fold more eotaxin, MIP-2, MIP-1, and
JE/MCP-1, respectively. In all cases, alterations in BAL cytokine
levels were associated with comparable alterations in chemokine mRNA
levels (Fig. 7). Thus, the enhanced
inflammatory response in OVA-sensitized and -challenged
IL-6-/- mice is associated with and potentially
mediated by the enhanced elaboration of these eosinophil and
mononuclear cell-regulating chemokines.
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Because the studies noted above demonstrate exaggerated
OVA-induced Th2 responses in IL-6-deficient mice, studies were
undertaken to determine whether exogenous IL-6 could inhibit
OVA-induced Th2 inflammation in otherwise normal animals. This was done
by comparing the OVA-induced responses in WT mice and mice in which
IL-6 is overexpressed in a lung/airway-specific fashion (CC10-IL-6
animals). In these experiments, the inflammatory effects of OVA
sensitization and challenge of WT mice were similar to those described
above. When similar experiments were undertaken with the CC10-IL-6
transgene (+) mice, a significant decrease in BAL cellularity was
noted. IL-6 also caused a marked decrease in BAL eosinophil recovery
and a marked decrease and increase in the percentage of BAL cells that
were eosinophils and macrophages, respectively. These effects were most
prominent 24 h after OVA challenge (data not shown). At this time
point, BAL eosinophil recovery was 5.5% of that seen in WT animals
(p < 0.001). Histology of the CC10-IL-6 mice
24 h after Ag challenge showed the scattered lymphoid aggregates
characteristic of these animals at baseline (31). In
contrast to the WT animals, no additional inflammation was noted (data
not shown). In addition, tissue eosinophilia was virtually undetectable
with the number of peribronchiolar eosinophils in the CC10-IL-6 mice
being 1% of the levels in identically manipulated WT mice
(p < 0.01). When viewed in combination, these
studies demonstrate that BAL and tissue inflammation and eosinophilia
are markedly diminished in OVA-sensitized and -challenged
CC10-IL-6 mice.
Th2 and Th2 regulating cytokines in CC10-IL-6 and WT mice
We next compared the production and expression of Th2 cytokines
and Th2 regulating cytokines in CC10-IL-6 and WT mice. OVA
sensitization and challenge caused a mild to moderate increase in IL-4,
IL-5, and IL-13 protein and mRNA in WT mice (Figs. 8 and 9).
IL-6 expression caused a significant decrease in the levels of these
cytokines and the mRNA transcripts that encode them (Figs. 8 and 9). It
also caused a significant decrease in the levels of BAL fluid
TGF-ß1 (Fig. 8) and did not significantly
increase the levels of IFN- protein or mRNA (data not shown). Thus,
IL-6 inhibits OVA-induced Th2 cytokine and
TGF-ß1 expression in this modeling system.
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Endothelial VCAM-1 staining was not detected in significant
quantities in WT animals or CC10-IL-6 mice that were unchallenged,
challenged with a saline aerosol, or challenged with Ag in the absence
of prior sensitization (data not shown). VCAM-1 staining of moderate
intensity was noted in blood vessels and capillaries 4 and 24 h
after challenge of sensitized WT animals. IL-6 markedly inhibited this
inductive response because CC10-IL-6 mice had no or minimal VCAM-1
expression at both time points (Fig. 10).
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C57BL/6 and BALB/c mice differ in their propensity to mount
specific types of T cell-mediated responses with the former having a
propensity toward Th1-dominated inflammation and the latter a
propensity toward Th2-dominated inflammation (42). To
determine whether IL-6 had similar inhibitory effects in the setting of
a genetic background that is predisposed to Th2 immunity, CC10-IL-6
transgene (+) mice on a BALB/c background and appropriate littermate
controls were sensitized and challenged as described above. In the
transgene (-) littermate controls, a brisk response was elicited with
an impressive increase in total BAL cellularity, an increase in BAL
eosinophilia, and the accumulation of significant levels of BAL fluid,
IL-4, IL-5, and IL-13. In the BALB/c CC10-IL-6 mice, total cellular
recovery, eosinophil recovery, percent BAL eosinophilia, and the levels
of BAL IL-4, IL-5, and IL-13 were all markedly diminished
(p < 0.01 for all comparisons) (Figs. 11 and 12). Thus, IL-6 is a potent inhibitor
of OVA-induced Th2 responses in BALB/c as well as C57BL/6
mice.
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Studies were also undertaken to determine whether the presence or
absence of IL-6 altered airway responsiveness to the nonspecific
agonist, methacholine, in C57BL/6 mice. This was done using barometric
whole-body plethysmography with measurements of
Penh, a documented indicator of airway
hyperresponsiveness after allergic sensitization in mice
(37). In keeping with prior observations and the mild
nature of our OVA challenge protocol, methacholine responsiveness was
only modestly enhanced by OVA sensitization and challenge in WT mice
(data not shown). In accord with prior studies with the CC10-IL-6 mice
(31) and the present demonstration that IL-6 inhibits
OVA-induced tissue inflammation, CC10-IL-6 mice manifest significantly
decreased levels of responsiveness to methacholine at baseline and
after OVA challenge when compared with WT controls (Fig. 13 and data not shown). At baseline,
the IL-6-/- mice manifest enhanced airway
responsiveness to methacholine. This approached did not achieve
statistical significance. However, statistically significant increases
in airway responsiveness were noted after OVA sensitization and
challenge (Fig. 13). When viewed in combination, these studies
demonstrate that the exaggerated expression of IL-6 diminishes, and a
deficiency in IL-6 augments airway hyperresponsiveness in
OVA-sensitized and -challenged C57BL/6 mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lymphocytes, eosinophils, and macrophages are characteristic of the
tissue inflammatory response in asthma and models of asthmatic
inflammation. An impressive volume of work has demonstrated that T
cells play an important role in these responses and that Th2
cell-derived IL-4, IL-5, and IL-13 are involved in the pathogenesis of
the increased IgE, eosinophilic infiltration, mucus metaplasia, and
physiologic dysregulation characteristic of the disorder (4, 5, 35, 43). A substantial body of literature has also demonstrated
that exaggerated IL-6 production is a feature of the asthmatic
diathesis (10, 14, 15, 16). However, the contribution of IL-6
to Th2 inflammation is controversial. Some reports suggest that IL-6
can augment Th2 inflammation. Specifically, Rincon et al. reported that
IL-6 polarized CD4+ cells to effector Th2 cells
by inducing IL-4 production (30), and Anguita demonstrated
that IL-6 contributed to IL-4 production in a murine model of Lyme
arthritis (19). In contrast, IL-6 has been shown to
inhibit Th2 inflammation and/or cytokine production in models of
Ag-induced arthritis and M. tuberculosis infection
(21, 22), and IL-6 has been shown to oppose the Th2
promoting effects of IL-10 in a Candida infection model
(13). Our studies address this controversy for the first
time in the airway. They support the inhibitory point of view by
demonstrating that IL-6 is a potent inhibitor of allergen-induced Th2
inflammation and tissue and BAL eosinophilia. In accord with these
findings, endogenous and exogenous IL-6 were also potent inhibitors of
aeroallergen-induced IL-4, IL-5, and IL-13 production. However, the
mechanisms of these inhibitory effects are not clear. They may be the
result of the ability of IL-6 to inhibit macrophage function, including
the production of pro-inflammatory cytokines such as IL-1 and TNF
(12, 24), induce the production of antiproteinases such as
the tissue inhibitors of metalloproteinases (44), and/or
stimulate the production of anti-inflammatory molecules such as
IL-1 receptor antagonist (45). The ability of IL-6 to
decrease endothelial cell VCAM-1 expression might also impede
eosinophil entry into tissues because VCAM-1-very late antigen (VLA)4
binding appears to be crucial for this response (46). In
addition, the exaggerated levels of TGF-ß1 in
OVA-challenged IL-6-/- mice could also shift
local tissue inflammation in a Th2 direction (41, 47) (see
below). However, it is clear that IL-6 does not inhibit Th2
inflammation by augmenting local Th1 responses because increases and
decreases in IFN- levels were not detected in challenged CC10-IL-6
and IL-6-/- mice, respectively.
Leukocyte chemotaxis is a important aspect of the pathogenesis of the
complex inflammatory responses in the airway. Studies of these
responses have demonstrated that multiple chemokines interact in a
well-orchestrated network(s) that coordinates the recruitment and/or
activation of the mononuclear cells and eosinophils seen in these
disorders (48, 49). Eotaxin, JE/MCP-1, MIP-1, and MIP-2
are well documented to contribute to these and similar tissue responses
(36, 48, 49, 50). Our studies demonstrate, for the first time,
that a deficiency in IL-6 results in the heightened elaboration of
these chemokine moieties after OVA challenge of the airway. It is
reasonable to speculate from these findings that the heightened levels
of these chemokines contribute to the increased tissue and BAL
inflammation seen in IL-6-/- mice. The
well-documented ability of IL-5 and eotaxin to synergize in regulating
eosinophil responses (50, 51) may also be important
because both are increased in OVA-challenged IL-6-deficient animals.
Recent studies from our laboratory and others have also demonstrated
that Th2 cytokines, in particular IL-13, are potent inducers of
chemokine elaboration by lung stromal cells (36, 52).
Thus, it is likely that the increased chemokine elaboration seen in
OVA-challenged IL-6-/- mice is due to the
heightened production of Th2 cytokines in these animals. However, we
cannot rule out the possibility that IL-6 has the ability to directly
inhibit cellular chemokine elaboration and that this inhibiting
mechanism is removed in the IL-6-/- mice. Thus,
additional investigation will be required to define the mechanism(s) of
the heightened chemokine response in
IL-6-/- mice.
The processes that determine whether airway inflammatory responses are transient or chronic are poorly understood. Chan-Yung and Malo (7, 53) attempted to define these processes using occupational asthma as an investigational paradigm. Their studies of the natural histories of these diseases highlighted four phases: 1) the period of initial exposure; 2) the period of sensitization during which airway inflammation probably develops; 3) the period where symptoms initially are appreciated; and 4) the period of possible resolution in which some patients lose their symptoms and physiologic hyperresponsiveness, whereas others go on to persistent asthma. These studies also suggest that disease progression is associated with continuing airway inflammation (7, 53). Resolution of tissue inflammation is a complex process that can occur as a result of the removal of the offending agent, active inhibition of inflammation by cytokines such as IL-10, and the apoptosis and clearance of infiltrating leukocytes. Thus, chronic inflammation can be the result of the persistence or repeated exposure to the inciting stimulus, inadequate inhibition of tissue inflammation, heightened target tissue sensitivity and/or responsiveness to proinflammatory signals or defects in apoptosis and/or leukocyte removal. Our studies demonstrate, for the first time, that IL-6 contributes to the active inhibition of inflammation in the airway. This finding has a number of important implications. First, it suggests that abnormalities in the production and/or effector functions of IL-6 can contribute to the generation, severity, and/or chronicity of asthma or other chronic inflammatory disorders of the airway. As a natural extension, polymorphisms of the IL-6 gene or the genes encoding the components of the IL-6 receptor and signal transduction pathway could contribute to the generation of the multiply diverse phenotypes seen in human asthma. In addition, the levels of IL-6, the soluble IL-6 receptor, and/or the presence of specific polymorphisms in the IL-6 system could serve as predictors of and biomarkers for chronic and/or severe asthmatic symptomatology.
IL-6 and TGF-ß1 interact in vivo and in vitro in a complex fashion. Studies from our laboratory and others have demonstrated that TGF-ß1 is a potent stimulator of IL-6 production and that TGF-ß1 and IL-6 can counterregulate each others’ effector functions under appropriate circumstances (39, 54, 55, 56). In addition, TGF-ß1 and IL-6 can have similar (57), antagonistic (58), and additive or synergistic (59, 60) biologic effector profiles. Because TGF-ß1 is a potent anti-inflammatory cytokine, studies were undertaken to define the role of TGF-ß1 in the inflammation-regulating effects of IL-6. To our surprise, an inverse relationship was noted with increased TGF-ß1 in BAL fluid from IL-6-/- mice and decreased TGF-ß1 in BAL fluid from CC10-IL-6 animals. Because TGF-ß1 can shift T cell responses in a Th2 direction (40, 41, 47, 61) and stimulate chemokine production by epithelial cells (62), it is tempting to speculate that IL-6 normally inhibits TGF-ß1 elaboration and that the heightened Th2 response in IL-6-/- mice is due, at least in part, to the heightened accumulation of TGF-ß1 in these animals. However, this attribution of cause and effect must be viewed with caution. Eosinophils are a major source of TGF-ß1 in the asthmatic lung (63). Thus, the increased TGF-ß1 in BAL fluid from IL-6-/- mice could be a consequence of the increased Th2 response and increased eosinophil influx in these animals and not the cause of this exaggerated reaction. Similarly, the decrease in BAL TGF-ß1 in the CC10-IL-6 mice may reflect the decreased eosinophil influx in these animals and not an ability of IL-6 to regulate cellular TGF-ß1 elaboration. In these circumstances, eosinophil-derived TGF-ß1 may be acting to augment, or in a counterregulatory fashion, to inhibit the local Th2 inflammatory response. Regardless, these findings clearly demonstrate that the anti-inflammatory effects of IL-6 do not appear to correlate with and are, therefore, not likely to be mediated solely by the effects of IL-6 on TGF-ß1 elaboration.
IL-6 is the prototype cytokine of the IL-6-type cytokine family. The members of this family (IL-6, IL-11, cardiotropin 1, oncostatin M, and ciliary neurotropic factor) share receptor components in their multimeric receptor complexes and have partially overlapping biologic effector profiles. These studies demonstrate that IL-6 inhibits Th2 inflammatory responses in the murine airway. Studies from our laboratory have demonstrated that IL-11 is a similarly impressive inhibitor of Th2 inflammation, eosinophilia, and cytokine elaboration in the airway (J. Wong and J. Elias, unpublished observation) and has protective effects in other injury systems (36, 64). When viewed in combination, these findings suggest that inhibition of Th2-dominated airway inflammation may be a general property of IL-6-type cytokines. They also suggest that IL-6-type cytokines and related compounds might be useful agents for the treatment of chronic Th2-dominated and other inflammatory responses in the airway.
In summary, these studies demonstrate that endogenous and exogenous
IL-6 inhibit aeroallergen-induced Th2-dominated tissue inflammation.
They also demonstrate that these IL-6-induced alterations in Th2
inflammation are not mediated by IFN- and can be appreciated in the
context of Th2-prone (BALB/c) and Th1-prone (C57BL/6) murine genetic
backgrounds. When these anti-inflammatory effects of IL-6 are
viewed in combination with the ability of IL-6 to stimulate the acute
phase response (8), antiproteinases (44),
anti-inflammatory cytokines (45), and tissue fibrosis
(32), it is clear that IL-6 is an important
anti-inflammatory, counterregulatory, and healing cytokine in the
airway.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jack A. Elias, Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, Department of Internal Medicine, 333 Cedar Street, 105 LCI, P.O. Box 208057, New Haven, CT 06520-8507.
3 Abbreviations used in this paper: WT, wild type; BAL, bronchoalveolar lavage; RPA, RNase protection assay; IHC, immunohistochemistry; Penh, enhanced pause; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein.
Received for publication February 4, 2000. Accepted for publication July 12, 2000.
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) and IL-6-/- (|og) mice were sensitized and
challenged with OVA as described in Materials and
Methods. The characteristics of the BAL fluids from these
animals were evaluated just before OVA challenge (0 h), or 4, 24, 48,
and 72 h and 7 days (d) after OVA challenge. Total cell recovery
(A), eosinophil recovery (B), the
percentage of BAL cells that were eosinophils (C), and
the percentage of BAL cells that were macrophages (D)
are illustrated (*, p < 0.01; **,
p < 0.001). The noted values represent the
mean ± SEM of a minimum of seven animals at each time
point.
) mice
were sensitized and challenged with OVA. The levels of IL-4, IL-13,
IL-5, and TGF-ß1 before and 24 h after aerosol
challenge, were evaluated by ELISA. The noted values represent the
mean ± SEM of a minimum of seven animals at each time point (*,
p < 0.01; **, p <
0.001).
