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Institute of Pharmacology, University of Messina School of Medicine, Messina, Italy;
Department of Experimental Pharmacology, University Federico II, Naples, Italy;
Department of Experimental and Clinical Medicine, University of Catanzaro School of Medicine, Catanzaro, Italy;
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Department of Biomorphology, University of Messina School of Medicine, Messina, Italy;
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Instituto di Richerche di Biologia Moleculare, P. Angeletti, Pomezia, Rome, Italy; and
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The William Harvey Research Institute, St. Bartholomew’s and The Royal London School of Medicine and Dentistry, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1 and possibly IL-6, contribute to
the extension of the inflammatory process. IL-6 is a multifunctional
cytokine that is produced and acts on a wide range of cells
(2, 3, 4). An enhanced formation of IL-6 has been reported in
patients with severe burns (5), after surgical operations
(6), during bacterial infections (7), in the
synovial fluid of patients with rheumatoid arthritis (8),
and in the cerebrospinal fluid of patients with bacterial meningitis
(9, 10). The role of IL-6 in the pathophysiology of
inflammation is still controversial. Recently, mice in which the gene
for IL-6 has been deleted (IL-6 knockout mice (IL-6KO mice)) have been
used to investigate the role of this cytokine in various models of
inflammation. In IL-6KO mice, the induction of acute phase proteins,
the weight loss, and the hypoglycemia caused by injection of turpentine
were dramatically reduced (11). The recruitment of
polymorphonuclear cells (PMNs) caused by injection of carrageenan in a
s.c. airpouch was also substantially reduced in IL-6KO mice compared
with that in wild-type (WT) mice (12). Most notably, the
arthritis (accumulation of PMNs in the knee joint and related tissue
damage) caused by collagen was abolished in IL-6KO mice
(13). IL-6-deficient mice are also protected against the
bone loss caused by estrogen depletion (14). All these
studies support a proinflammatory role of IL-6. There are, however,
other studies that do not document a proinflammatory role of IL-6 or
even suggest that IL-6 may be an anti-inflammatory cytokine. For
instance, IL-6KO mice treated with LPS produced 3-fold more TNF-
than their WT controls suggesting that endogenous IL-6 may be important
in the control of LPS-induced TNF- synthesis. Similar to WT
controls, LPS-treated IL-6 deficient mice develop anorexia, weight
loss, and hypoglycemia, suggesting that IL-6 is not required to
generate and inflammatory response to LPS and may, in fact, exert a
protective effect during acute inflammation (15). In
addition, IL-6 is not necessary for the suppression of the synthesis of
proteoglycan caused by IL-1 and leukemia inhibitory factor
(16). Most notably, the cartilage destruction caused by
injection of zymosan into the knee joint is significantly enhanced in
IL-6KO mice, while injection of IL-6 (into the knee joint) protects WT
mice against this zymosan-induced cartilage degradation. Thus, it
appears that the role of IL-6 in acute inflammation depends on the
stimulus and/or the model of inflammation used. Injection of carrageenan into the pleural space leads to pleurisy, PMN infiltration, and lung injury. Models of carrageenan-induced pleurisy have been widely employed to investigate the pathophysiology of acute inflammation and to evaluate the efficacy of drugs in inflammation. Interestingly, IL-6KO mice have not been used to elucidate whether IL-6 plays a pro- or an anti-inflammatory role in this model. This is surprising, as the injection of carrageenan leads to a rapid and substantial rise in IL-6, which is maximal after 4 h, but IL-6 remains elevated for up to 16 h after injection of carrageenan. In this study we have investigated the role of IL-6 in a model of carrageenan-induced pleurisy using IL-6KO mice and IL-6WT mice. To characterize the role of IL-6 in this model of acute inflammation, we have determined the following end points of the inflammatory response in IL-6KO mice and in the corresponding WT mice: 1) exudate formation, 2) PMN infiltration, 3) peroxynitrite formation (immunohistochemistry), 4) activation of the nuclear enzyme poly(A)DP-ribose polymerase (PARP), 5) expression of cyclo-oxygenase-2 (COX-2) protein (immunohistochemistry) and activity, 6) formation of leukotriene B4, 7) expression of the inducible NO synthase (iNOS) protein (immunohistochemistry) and activity, 8) lipid peroxidation, and 9) lung injury. In addition, we have investigated the effects of the systemic administration (pretreatment) of an mAb against IL-6 on the above parameters of inflammation in WT mice subjected to carrageenan-induced pleurisy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male IL-6KO and IL-6WT mice (20–22 g) were used to assess the role of IL-6 in the pathogenesis of carrageenan-induced pleurisy. All animals were allowed access to food and water ad libitum. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192) as well as with the European Economic Community regulations (O.J. of E.C. L 358/1 12/18/1986).
Carrageenan-induced pleurisy
Mice were anesthetized with isoflurane and submitted to a skin
incision at the level of the left sixth intercostal space. The
underlying muscle was dissected, and saline (0.2 ml) or saline
containing 1% 
-carrageenan (0.2 ml) was injected into the pleural
cavity. The skin incision was closed with a suture, and the animals
were allowed to recover. At 4 h after the injection of
carrageenan, the animals were euthanized by inhalation of
CO2. The chest was carefully opened, and the
pleural cavity was rinsed with 2 ml of saline solution containing
heparin (5 U/ml) and indomethacin (10 µg/ml). The exudate and washing
solution were removed by aspiration, and the total volume was measured.
Any exudate that was contaminated with blood was discarded. The amount
of exudate was calculated by subtracting the volume injected (2 ml)
from the total volume recovered. The leukocytes in the exudate were
suspended in PBS and counted with an optical microscope in a Burker’s
chamber after vital trypan blue staining. In a second group of
experiments, pleurisy was induced in IL-6WT mice treated with
anti-mouse IL-6 Ab (5 µg/day; Genzyme, Cambridge, MA) injected
i.p. at 24 and 1 h before carrageenan administration. The
dose of the IL-6 Ab used here has been demonstrated to abolish the
effects of endogenous IL-6 (10, 17).
Cell culture
Resident pleural cells macrophages were collected from mice
4 h after the carrageenan injection as previously described
(3). The cells (106/ml), which were
mainly macrophages (
70%), were cultured in DMEM supplemented with
L-glutamine (3.5 mM), penicillin (50 U/ml), streptomycin
(50 µg/ml), and heparin sodium (10 U/ml) in 12-well plates for 2
h and were allowed cells to adhere at 37°C in a humidified 5%
CO2 incubator. Nonadherent cells were removed by
rinsing the plates three times with 5% dextrose water. After removing
nonadherent cells (10%), adherent macrophages were scraped for the
measurement of DNA strand breaks and cellular
NAD+. Mitochondrial respiration and peroxynitrite
formation were measured in the adherent cells in the subsequent 1-h
period.
Measurement of nitrite/nitrate
Nitrite plus nitrate (NOx) production, an indicator of NO synthesis, was measured in the supernatant samples as previously described (18). Briefly, the nitrate in the supernatant was first reduced to nitrite by incubation with nitrate reductase (670 mU/ml) and NADPH (160 µM) at room temperature for 3 h. The nitrite concentration in the samples was then measured by the Griess reaction, by adding 100 µl of Griess reagent (0.1% naphthylethylenediamide dihydrochloride in H2O and 1% sulfanilamide in 5% concentrated H2PO4; 1/1, v/v) to 100-µl samples. The OD at 550 nm (OD550) was measured using an ELISA microplate reader (SLT-Labinstruments, Salzburg, Austria). Nitrate concentrations were calculated by comparison with OD550 of standard solutions of DMEM.
Determination of NOS activity
The calcium-independent conversion of L-arginine to L-citrulline in the homogenates of lungs (obtained 4 h after carrageenan treatment) served as an indicator of iNOS activity (18). Lungs were scraped into a homogenization buffer composed of 50 mM Tris-HCl, 0.1 mM EDTA, and 1 mM PMSF (pH 7.4) and homogenized in the buffer on ice using a tissue homogenizer. Conversion of L-[3H]arginine to L-[3H]citrulline was measured in the homogenates as previously described (3). Briefly, homogenates (30 µl) were incubated in the presence of L-[3H]arginine (10 µM, 5 kBq/tube), NADPH (1 mM), calmodulin (30 nM), tetrahydrobiopterin (5 µM), and EGTA (2 mM) for 20 min at 22°C. Reactions were stopped by dilution with 0.5 ml of ice-cold HEPES buffer (pH 5.5) containing EGTA (2 mM) and EDTA (2 mM). Reaction mixtures were applied to Dowex 50W (Na+ form) columns and the eluted L-[3H]citrulline activity was measured by a Beckman scintillation counter (Palo Alto, CA).
Measurement of peroxynitrite-induced oxidation of dihydrorhodamine 123
The formation of peroxynitrite was measured by the peroxynitrite-dependent oxidation of dihydrorhodamine 123 to rhodamine 123, as previously described (18). Cells were rinsed with PBS, and the medium was then replaced with PBS containing 5 µM dihydrorhodamine 123. After a 60-min incubation at 37°C, the fluorescence of rhodamine 123 was measured using a fluorometer at an excitation wavelength of 500 nm and an emission wavelength of 536 nm (slit widths, 2.5 and 3.0 nm, respectively). Thus, this method is an indirect measurement of peroxynitrite production because also other oxidant species can induce oxidation of dihydrorhodamine 123.
Measurement of mitochondrial respiration
Cell respiration was assessed by measuring the mitochondrial-dependent reduction of MTT to formazan (18). Cells in 96-well plates were incubated at 37°C with MTT (0.2 mg/ml) for 1 h. Culture medium was removed by aspiration, and the cells were solubilized in DMSO (100 µl). The extent of reduction of MTT to formazan within cells was quantified by the measurement of OD550. As previously discussed (18), the measurement of MTT reduction appears to be mainly by the mitochondrial complexes I and II, but may also involve NADH- and NADPH-dependent energetic processes that occur outside the mitochondrial inner membrane. Thus, this method cannot be used to separate the effects of free radicals, oxidants, or other factors on the individual enzymes in the mitochondrial respiratory chain, but is useful to monitor changes in the general energetic status of the cells.
Determination of DNA single-strand breaks
The formation of DNA strand breaks in dsDNA was determined by the alkaline unwinding methods as previously described (18). Cells in 12-well plates were scraped into 0.2 ml of solution A buffer (250 mM myo-inositol, 10 mM NaH2PO3, and 1 mM MgCl2, pH 7.2). The cell lysate was then transferred into plastic tubes designated T (maximum fluorescence), P (fluorescence in sample used to estimate extent of DNA unwinding), or B (background fluorescence). To each tube, 0.2 ml of solution B (alkaline lysis solution: 10 mM NaOH, 9 M urea, 2.5 mM EDTA, and 0.1% SDS) was added and incubated at 4°C for 10 min to allow cell lysis and chromatin disruption; 0.1 ml each of solutions C (0.45 vol of solution B in 0.2 N NaOH) and D (0.4 vol of solution B in 0.2 N NaOH) were then added to the P and B tubes, and 0.1 ml of solution E (neutralizing solution: 1 M glucose and 14 mM ME) was added to the T tubes before solutions C and D were added. From this point onward, all incubations were conducted in the dark. A 30-min incubation period at 0°C was then allowed, during which the alkali diffused into the viscous lysate. As the neutralizing solution, solution E, was added to the T tubes before addition of the alkaline solutions C and D, the DNA in the T tubes was never exposed to a denaturing pH. At the end of the 30-min incubation, the contents of the B tubes were sonicated for 30 s to ensure rapid denaturation of DNA in the alkaline solution. All tubes were then incubated at 15°C for 10 min. Denaturation was stopped by chilling to 0°C and adding 0.4 ml of solution E to the P and B tubes. Then, 1.5 ml of solution F (6.7 µg/ml ethidium bromide in 13.3 mM NaOH) was added to all the tubes, and fluorescence (excitation, 520 nm; emission, 590 nm) was measured by a fluorometer. Under the conditions used, in which ethidium bromide binds preferentially to dsDNA, the percentage of dsDNA (D) may be determined using the equation: % D = 100 X [F(P) - F(B)]/[F(T) - F (B)], where F(P) is the fluorescence of the sample, F(B) is the background fluorescence, i.e., fluorescence due to all cell components other than dsDNA, and F(T) is the maximum fluorescence.
Measurement of cellular NAD+ levels
Cells in 12-well plates were extracted in 0.25 ml of 0.5 N HClO4 scraped, neutralized with 3 M KOH, and centrifuged for 2 min at 10,000 x g. The supernatant was assayed for NAD+ using a modification of the colorimetric method (18) in which NADH produced by enzymatic cycling with alcohol dehydrogenase reduces MTT to formazan through the intermediation of phenazine methasulfate. The rate of MTT reduction is proportional to the concentration of the coenzyme. The reaction mixture contained 10 µl of a solution of 2.5 mg/ml MTT, 20 µl of a solution of 4 mg/ml phenazine methosulfate, 10 µl of a solution of 0.6 mg/ml alcohol dehydrogenase (300 U/mg), and 190 µl of 0.065 M glycyl-glycine buffer, pH 7.4, that contained 0.1 M nicotinamide and 0.5 M ethanol. The mixture was warmed to 37°C for 10 min, and the reaction was started by the addiction of 20 µl of the sample. The rate of increase in absorbance was read immediately after the addition of NAD+ samples and after 10- and 20-min incubation at 37°C against blank at 560 nm in the ELISA microplate reader (SLT-Labinstruments).
Measurement of cytokines
TNF- and IL-1ß levels were evaluated in the exudate at
4 h after the induction of pleurisy by carrageenan injection. The
assay was conducted by using a colorimetric, commercial kit
(Calbiochem-Novabiochem, La Jolla, CA). The ELISA has a lower detection
limit of 30 pg/ml.
Immunohistochemistry of iNOS
Lung biopsies were taken 4 h after the induction of pleurisy by carrageenan injection. The lungs were perfused for 15 min with fresh 3.5% cacodylate-buffered paraformaldehyde, and cryostat sections were prepared from the fixed lung tissue. Endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 3% normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min each with avidin and biotin (DBA, Milan, Italy). The sections were then incubated overnight with primary anti-iNOS Ab or control solutions as previously described (19). Controls included buffer alone or nonspecific purified rabbit IgG. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (DBA).
Measurement of PGE2 in the pleural exudate
The amount of PGE2 present in the pleural fluid was measured by RIA without prior extraction or purification (20).
Assessment of COX activity
Lungs were obtained at 4 h after the induction of pleurisy
by carrageenan injection. The material was homogenized at 4°C in a
buffer containing the following protease inhibitors in a ratio of 5/1
(v/w). The protein concentration in the homogenates was measured by the
Bradford assay (21), with BSA used as the standard.
Homogenates were incubated at 37°C for 30 min in the presence of
excess arachidonic acid (30 µM). The samples were boiled and
centrifuged at 10,000 x g for min. The concentration
of 6- keto-PGF1 present in the supernatant
was measured by RIA as previously described (22).
Immunohistochemical localization of COX-1 and COX-2
Lung biopsies were fixed in 10% buffered formalin, and 8-µm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Nonspecific binding was minimized by incubating the section in 2% normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin (DBA). The sections were then incubated overnight with a 1/500 dilution of the primary anti-COX-1 or anti-COX-2 Ab (DBA) or with control solutions. Controls included buffer alone or nonspecific purified rabbit IgG. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase (DBA).
Measurement of LTB4
LTB4 levels were evaluated in the exudate at 4 h after the induction of pleurisy by carrageenan injection. The assay was conducted by using a colorimetric commercial kit (Calbiochem-Novabiochem).
Immunohistochemical localization of nitrotyrosine
Tyrosine nitration, an index of the nitrosylation of proteins by peroxynitrite and/or oxygen-derived free radicals, was determined by immunohistochemistry as previously described (18). At the end of the experiment, the relevant organs were fixed in 10% buffered formaldehyde, and 8-µm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min each with avidin and biotin. The sections were then incubated overnight with a 1/1000 dilution of primary anti-nitrotyrosine Ab or with control solutions. Controls included buffer alone or nonspecific purified rabbit IgG. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (DBA).
Immunohistochemical localization of PARP
At 4 h after carrageenan injection, lung tissues were fixed in 10% buffered formalin, and 8-µm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin (DBA). The sections were then incubated overnight with a 1/500 dilution of primary anti-PARP Ab (DBA) or with control solutions. Controls included buffer alone or nonspecific purified rabbit IgG. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase (DBA).
Light microscopy
Lung biopsies were taken at 4 h after injection of carrageenan. The biopsies were fixed for 1 wk in buffered formaldehyde solution (10% in PBS) at room temperature, dehydrated by graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Tissue sections (thickness, 7 µm) were deparaffinized with xylene, stained with trichromic Van Gieson, and studied using light microscopy (Dialux 22, Leitz, Rockleigh, NJ).
Determination of myeloperoxidase activity
Myeloperoxidase (MPO) activity, an indicator of PMN accumulation, was determined as previously described (23). At 4 h after intrapleural injection of carrageenan, lung tissues were obtained and weighed. Each piece of tissue was homogenized in a solution containing 0.5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 x g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 µmol of peroxide min at 37°C and was expressed in milliunits per gram weight of wet tissue.
Determination of malondialdehyde (MDA) levels
The levels of MDA in lung tissue were determined as an indicator of lipid peroxidation (24). Lung tissue, collected at the specified time, was homogenized in 1.15% KCl solution. An aliquot (100 µl) of the homogenate was added to a reaction mixture containing 200 µl of 8.1% SDS, 1500 µl of 20% acetic acid (pH 3.5), 1500 µl of 0.8% thiobarbituric acid, and 700 µl of distilled water. Samples were then boiled for 1 h at 95°C and centrifuged at 3000 x g for 10 min. The absorbance of the supernatant was measured by spectrophotometry at 650 nm.
Materials
Biotin blocking kit, biotin-conjugated goat anti-rabbit IgG, primary anti-nitrotyrosine, anti-COX-2, anti-poly(A)DP-ribose Ab, and avidin-biotin peroxidase complex were obtained from DBA. All other reagents and compounds used were obtained from Sigma (Milan, Italy).
Data analysis
All values in the figures and text are expressed as the mean ± SEM of n observations. For the in vitro studies, data represent the number of wells studied (six to nine wells from two or three independent experiments). For the in vivo studies, n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. p < 0.05 was considered significant
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All WT mice that had received carrageenan developed acute
pleurisy, producing 0.9 ± 0.02 ml of turbid exudate (Fig. 1
a). Compared with the number
of cells collected from the pleural space of sham-operated mice
(0.9 ± 0.02 x 106/mice; Fig. 1b), injection of carrageenan induced a significant increase
in the number of PMNs (53 ± 1.5 x
106/mice; Fig. 1b). Pretreatment of
IL-6WT mice with anti-IL-6 Ab as well as IL-6KO mice showed a
significant attenuation of the pleural exudate as well as the number of
PMNs within the exudate (Fig. 1, a and b). No
significant exudate and no significant increase in the number of PMNs
were observed in the pleural cavity of sham-operated mice.
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The levels of TNF- and IL-1ß were significantly elevated in
the exudate from IL-6WT mice at 4 h after carrageenan
administration. In contrast, the levels of these cytokines were
significantly lower in IL-6KO mice and IL-6WT mice treated with an Ab
against IL-6 (Fig. 2). No significant
cytokine increased was observed in the exudate of sham-operated
mice.
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All IL-6WT mice that were treated with carrageenan exhibited a
substantial increase in the activities of MPO and MDA in the lung (Fig. 3, a and b).
Pretreatment of IL-6WT mice with anti-IL-6 Ab as well as IL-6KO
mice showed a significant attenuation of the increases in MPO and MDA
caused by carrageenan in the lung (Fig. 3, a and
b). There was no increase in either MPO activity or MDA
level in sham-operated animals. Histological examination of lung
sections of IL-6WT mice treated with carrageenan showed edema, tissue
injury, as well as infiltration of the tissue with PMNs, lymphocytes,
and plasma cells (Fig. 4a).
Pretreatment of IL-6WT mice with anti-IL-6 Ab as well as IL-6KO
mice showed a significant reduction of the lung injury as well as the
infiltration of the tissue with white blood cells (Fig. 4, b
and c). No histological alteration was found in
sham-operated mice (data not shown).
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The levels of NOx were significantly (p <
0.01) increased in the exudate from carrageenan-treated WT mice
(141 ± 9 vs 11 ± 2 nmol/sham mice; Fig. 5a). In contrast, the levels
of NOx were significantly lower in the exudate of carrageenan-treated
IL-6KO and carrageenan-treated IL-6WT mice treated with the
anti-IL-6 Ab (Fig. 5a). In the lungs obtained from
animals subjected to carrageenan-induced pleurisy, a significant
increase in iNOS activity was detected at 4 h (Fig. 5b). The iNOS activity was significantly
(p < 0.01) lower in IL-6KO mice and IL-6WT
mice treated with the anti-IL-6 Ab (Fig. 5b).
Immunohistochemical analysis of lung sections obtained from IL-6WT mice
treated with carrageenan revealed positive staining for iNOS, which was
primarily localized in alveolar macrophages (Fig. 6a). In contrast, no positive
iNOS staining was found in the lungs of carrageenan-treated IL-6KO mice
and IL-6WT mice treated with the anti-IL-6 Ab (Fig. 6, b
and c). Staining was absent in control tissue (data not
shown).
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The COX activity in carrageenan-induced pleural exudate and lung
homogenates was assessed by measuring the increase in the formation of
PGE2 in the exudate. The amounts of
PGE2 found in the pleural exudate of
carrageenan-treated WT mice was 475 ± 19 pg/mice
(n = 6). The amounts of PGE2 were
significantly lower in the exudate obtained from IL-6KO mice as well as
from IL-6WT mice treated with the anti-IL-6 Ab (Fig. 7a). In lungs from
carrageenan-treated WT mice, the amount of
6-keto-PGF1 was 271 ± 30 pg/mg/tissue
(Fig. 7b). The amount of
6-keto-PGF1 was significantly reduced in the
lungs from carrageenan-treated IL-6KO mice as well as from IL-6WT mice
treated with the anti-IL-6 Ab (Fig. 7b).
Immunohistochemical analysis of lung sections obtained from IL-6WT mice
treated with carrageenan revealed a positive staining for COX-2, which
was primarily localized in alveolar macrophages (Fig. 8a). In contrast, no positive
COX-2 staining was found in the lungs of carrageenan-treated IL-6KO
mice and IL-6WT mice treated with the anti-IL-6 Ab (Fig. 8, b and c). Staining was absent in tissue obtained
from sham-operated control animals (data not shown).
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LTB4 production
The levels of LTB4 were significantly
elevated in the exudate from IL-6WT mice at 4 h after carrageenan
administration. In contrast, the levels of LTB4
were significantly (p < 0.05) lower in IL-6KO
mice and IL-6WT mice treated with an Ab against IL-6 (Fig. 9).
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Immunohistochemical analysis of lung sections obtained from IL-6WT
mice treated with carrageenan revealed positive staining for
nitrotyrosine, which was primarily localized in alveolar macrophages
and airway epithelial cells (Fig. 10a). In contrast, no
positive nitrotyrosine staining was found in the lungs of
carrageenan-treated IL-6KO mice and IL-6WT mice treated with the
anti-IL-6 Ab (Fig. 10, b and c).
Immunohistochemical analysis of lung sections obtained from IL-6WT mice
treated with carrageenan revealed positive staining for PARP (Fig. 11a). In contrast, no
positive PARP staining was found in the lungs of carrageenan-treated
IL-6KO mice or in IL-6WT mice treated with the anti-IL-6 Ab (Fig. 12, b and c).
Note that there was no staining for either nitrotyrosine or PARP in
lungs obtained from sham-operated mice (data not shown).
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Compared with the supernatant of macrophages collected from the
pleural cavity of sham-operated animals, the supernatant of
macrophages obtained from carrageenan-treated IL-6WT mice showed
a significant increase in the concentration of NOx (Fig. 13a). This was associated
with a significant increase in iNOS activity in these cells (Fig. 13b). The amount of NOx found in the supernatant of
macrophages obtained from IL-6WT mice that had been pretreated with
anti-IL-6 Ab or from IL-6KO mice was significantly lower.
Similarly, the iNOS activity measured in the macrophages from
carrageenan-treated IL-6KO mice and IL-6WT mice pretreated with
anti-IL-6 Ab was significantly lower (Fig. 13, a and
b). Compared with the supernatant of macrophages collected
from the pleural cavity of sham-operated animals, the supernatant of
macrophages obtained from carrageenan-treated IL-6WT mice showed a
significant increase in the concentration of peroxynitrite (Fig. 13c). This was associated with a significant increase in the
occurrence of single-strand breaks in the DNA (Fig. 13d), a
reduction in mitochondrial respiration (Fig. 13e), as well
as a significant fall in the intracellular levels of NAD (Fig. 13f) in these cells. Macrophages obtained from IL-6WT mice
that had been pretreated with anti-IL-6 Ab or from IL-6KO mice show
an attenuation of the formation of peroxynitrite (Fig. 13c)
as well as the associated DNA damage (Fig. 13d), impairment
of mitochondrial respiration (Fig. 13e), as well as a fall
in NAD levels (Fig. 13f).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, and IL-6. Using mice in which
the gene for IL-6 was deleted (IL-6KO mice), we have investigated the
role of IL-6 in the inflammation associated with the injection of
carrageenan in the pleural cavity. We demonstrate that 1) the
development of carrageenan-induced pleurisy, 2) the infiltration of the
lung with PMNs (histology and MPO activity), 3) the degree of lipid
peroxidation in the lung, and 4) the degree of lung injury (histology)
caused by injection of carrageenan are significantly attenuated in
IL-6KO mice (compared with those in wild-type mice treated with
carrageenan). All these findings suggest that endogenous IL-6 augments
the inflammation associated with carrageenan-induced pleurisy. To
support the hypothesis that endogenous IL-6 plays a proinflammatory
role in the model of inflammation used here, we have also pretreated
wild-type mice with an Ab against IL-6 and subsequently subjected these
animals to injection of carrageenan into the pleural space.
Interestingly, pretreatment of wild-type mice with an Ab against IL-6
also attenuated the development of carrageenan-induced pleurisy, the
infiltration of the lung with PMNs, the degree of lipid peroxidation,
and the degree of lung injury caused by injection of carrageenan. Taken
together, these two studies strongly support the view that IL-6 plays a
proinflammatory role in carrageenan-induced pleurisy in the rat. What,
then, is the mechanism by which IL-6 augments the inflammation caused
by injection of carrageenan into the pleural cavity of the rat?
There is evidence that the proinflammatory cytokines TNF- and IL-1
help to propagate the extension of a local or systemic inflammatory
process (11, 12, 25). We confirm here that the
inflammatory process caused by injection of carrageenan into the
pleural cavity leads to a substantial increase in the levels of both
TNF- and IL-1 in the exudate. Interestingly, the levels of these two
proinflammatory cytokines are significantly lower in the exudate
obtained from animals that are unable to produce endogenous IL-6
(IL-6KO mice) or from WT mice in which the effects of endogenous IL-6
have been attenuated by an Ab against this cytokine. These findings
suggest that in the presence of endogenous IL-6, the degree of
inflammation and, hence, the formation of TNF- and IL-1 are
significantly enhanced (e.g., by positive feedback).
There is a large amount of evidence that the production of ROS such as
hydrogen peroxide, superoxide, and hydroxyl radicals at the site of
inflammation contributes to tissue damage (18, 26, 27, 28, 29).
Inhibitors of NOS activity reduce the development of
carrageenan-induced inflammation and support a role for NO in the
pathophysiology associated with this model of inflammation (17, 29, 30, 31, 32). We demonstrate here that the formation of nitrite and
nitrate (metabolites of NO in water) as well as the induction of iNOS
protein in macrophages and airway epithelial cells caused by injection
of carrageenan into the pleural cavity are reduced in lungs of IL-6KO
mice as well as in WT mice pretreated with an Ab against IL-6. This
finding suggests that endogenous IL-6 amplifies the induction of iNOS
caused by carrageenan in the lung. The induction of iNOS caused, e.g.,
by injection of endotoxin in rodents in vivo is mediated by endogenous
TNF-, as polyclonal Abs against this cytokine abolish the induction
of iNOS caused by endotoxin in the rat (33). Like TNF-,
endogenous IL-1 also plays an important role in the induction of iNOS,
as the endogenous IL-1R antagonist also reduces the degree of iNOS
induction caused by LPS in rodents (34). As the levels of
TNF- and IL-1 are significantly lower in the exudate obtained from
IL-6KO mice and from WT mice pretreated with an Ab against IL-6, we
propose that the attenuation of the induction of iNOS protein and
activity observed in mice that are unable to produce endogenous IL-6
(IL-6KO mice) are secondary to reduced formation of endogenous TNF-
and IL-1. Like iNOS, the expression of COX-2 is also mediated by
TNF- and IL-1. As the levels of TNF- and IL-1 as well as the
induction of iNOS are significantly lower in the exudate obtained from
IL-6KO mice (and from WT mice pretreated with an Ab against IL-6), it
is not surprising that the degree of COX-2 induction is also
significantly attenuated in macrophages located in the lungs of IL-6KO
mice subjected to carrageenan-induced pleurisy. There is good evidence
in this and other models of inflammation that enhanced formation of
prostanoids following the induction of COX-2 contributes to the
pathophysiology of local inflammation (35, 36) and that
selective inhibitors of COX-2 exert potent anti-inflammatory
effects (37, 38, 39). We demonstrate here that the increase in
the levels of PGE2 caused by injection of
carrageenan into the pleural cavity is reduced in the exudate of IL-6KO
mice as well as in WT mice pretreated with an Ab against IL-6. The
enhanced formation of PGE2 is secondary to the
expression of COX-2 protein, as 1) there was no increase in the
expression of COX-1 protein (detected by immunohistochemistry) after
carrageenan injection; and 2) selective inhibitors of COX-2 activity,
including NS-398 (nimesulide) and SC-58125 (Celecoxib), abolished the
increase in PGE2 caused by injection of
carrageenan into the pleural space (38) Thus, we propose
that IL-6 amplifies the expression of COX-2 protein and activity caused
by injection of carrageenan in the lung and that the subsequent
enhanced formation of PGE2 (and other COX-2
metabolites) contributes to the observed inflammatory process. This
conclusion is supported by the previous finding that selective
inhibitors of COX-2 activity reduced the inflammation caused by
carrageenan in the rat (37, 38, 39).
In addition to PGs and NO, peroxynitrite is also generated in carrageenan-induced inflammation (18, 27, 29). The biological activity and decomposition of peroxynitrite are very much dependent on the cellular or chemical environment (presence of proteins, thiols, and glucose; ratio of NO and superoxide; carbon dioxide levels; and other factors), and these factors influence its toxic potential (40, 41, 42). We demonstrate here that injection of carrageenan into the pleural cavity of WT mice leads to a substantial increase in the degree of nitrosylation of proteins in the lung. In contrast, the degree of staining for nitrotyrosine was significantly reduced in IL-6KO mice or in WT mice pretreated with an Ab against IL-6. Nitrotyrosine formation along with its detection by immunostaining were initially proposed as relatively specific markers for the detection of the endogenous formation (footprint) of peroxynitrite (43). There is, however, recent evidence that certain other reactions can also induce tyrosine nitration; e.g., the reaction of nitrite with hypoclorous acid and the reaction of myeloperoxidase with hydrogen peroxide can lead to the formation of nitrotyrosine (44). Increased nitrotyrosine staining is considered, therefore, an indication of increased nitrosative stress rather than a specific marker of the generation of peroxynitrite. Thus, our results suggest that the degree of nitrosative stress caused by injection of carrageenan is reduced in lungs from animals that are unable to produce endogenous IL-6 or those in which the effects of endogenous IL-6 have been attenuated/abolished by an Ab against this cytokine.
ROS and peroxynitrite produce cellular injury and necrosis via several mechanisms, including peroxidation of membrane lipids, protein denaturation, and DNA damage. ROS produce strand breaks in DNA, which triggers energy-consuming DNA repair mechanisms and activates the nuclear enzyme PARS, resulting in the depletion of its substrate NAD in vitro and a reduction in the rate of glycolysis. As NAD functions as a cofactor in glycolysis and the tricarboxylic acid cycle, NAD depletion leads to a rapid fall in intracellular ATP. This process has been termed the PARS suicide hypothesis (45, 46). There is recent evidence that the activation of PARS may also play an important role in inflammation (45, 46, 47, 48). We also demonstrate here that the increase in PARS activity caused by carrageenan in the lung is attenuated in lungs of IL-6KO mice as well as in lungs of WT mice pretreated with an Ab against IL-6.
In conclusion, this study demonstrates that the degree of inflammation
caused by injection of carrageenan in the pleural cavity of the mouse
is significantly attenuated in IL-6KO mice. Similarly, pretreatment of
WT mice with an Ab against IL-6 reduced the inflammatory response
caused by subsequent injection of carrageenan. These findings support
the view that endogenous IL-6 contributes to the extension of
inflammation in the model of carrageenan-induced pleurisy used
here. The mechanisms of the proinflammatory effect of IL-6 are not
entirely clear. It appears that IL-6 augments (positive feedback; Fig. 12
) the formation of other proinflammatory cytokines, such as TNF-
and IL-1, which, in turn, may augment the recruitment of neutrophils,
the expression of iNOS and COX-2 protein and activity, and ultimately
the degree of peroxynitrite formation and tissue injury. In addition,
IL-6 appears to enhance the formation of LTB4,
which, in turn, may contribute to the recruitment of PMNs. Finally, our
findings suggest that interventions that may reduce the generation or
the effects of IL-6 may be useful in conditions associated with local
or systemic inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Salvatore Cuzzocrea, Institute of Pharmacology, University of Messina School of Medicine, Piazza XX Settembre No. 4, 98123 Messina, Italy. E-mail address:
3 Abbreviations used in this paper: ROS, reactive oxygen species; IL-6KO, IL-6 knockout mice; PMN, polymorphonuclear cells; IL-6WT, IL-6 wild-type mice; LTB4, leukotriene B4; PARP, poly(A)DP-ribose polymerase; COS-2, cyclo-oxygenase protein-2; iNOS, inducible NO synthase; NOx, nitrite plus nitrate; MPO, myeloperoxidase; MDA, malondialdehyde.
Received for publication May 14, 1999. Accepted for publication July 23, 1999.
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B. NO, in turn, combined with superoxide to yield
peroxynitrite. Hydroxyl radical and peroxynitrite induced cellular
injury. Part of the injury was related to the development of DNA
single-strand breakage, with consequent activation of PARS, leading to
cellular dysfunction and morphological alteration. NO may directly
increase the catalytic activity of the inducible isoform of COX
(COX-2), leading to enhanced production of proinflammatory PG
metabolites. In this system, the inhibition of IL-6 may produce 1)
regulation of cytokine release, 2) modulation of PMN infiltration, 3)
inhibition of the activation of NF-