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From the Department of Pathology, Medical College of Wisconsin, Milwaukee, Wisconsin
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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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We have demonstrated in vitro the priming effects of in vivo exposure to cigarette smoke using neutrophils purified from whole blood of nonsmokers and a low molecular weight polyphenol-rich fraction, referred to as cigarette smoke condensate (CSC), extracted from cigarette smoke.4 In cigarette smoking-associated diseases, smoking cessation should remove the priming agent thereby reducing inflammation and slowing the disease process. This, however, is not always the case. In some patients with COPD the disease continues long after smoking has stopped.5 Airway inflammation, marked by a significant increase in the percentage of neutrophils in the sputum and cells containing IL-8, has been demonstrated in COPD patients at least 1 year after smoking cessation.6 One hypothesis to explain ongoing inflammation in these patients would be the presence of anti-idiotypic antibodies with activity similar to agents in tobacco, like CSC, that could function to continuously prime neutrophils and thereby contribute to the continuation of the inflammatory process. To develop an in vitro model to test this hypothesis, we generated a mouse monoclonal tobacco anti-idiotypic antibody (TAIA) reactive with rabbit antibodies to polyphenols in tobacco that is functionally similar to CSC.7 Both CSC and TAIA prime neutrophils to show a significant increase in fMLP receptors and expression of the integrin CD11b/18 on the cell membrane. Neutrophils primed with CSC and TAIA also have significantly increased oxidative burst activity and release of elastase after stimulation with fMLP compared with unprimed controls. Neutrophil priming by CSC is abrogated in the presence of SB203580, an inhibitor of MAPK p38 activation and phosphorylation, indicating a role for activation of MAPK pathways in this process.
The results presented in this in vitro study support the hypothesis that TAIA, which is functionally similar to CSC, may contribute to driving progression of COPD long after smoking has stopped. This hypothesis is novel and carries with it the implication of a heretofore unrecognized autoimmune component of smoking-associated diseases manifested through production of anti-idiotypic antibodies with tobacco-like activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cigarette smoke condensate was obtained from the Phillip Morris Company, Richmond, Virginia. Twenty-five g of condensate were extracted with 400 ml of phosphate-buffered saline (PBS) for 18 hours at 50 to 56°C. During the early hours of extraction, pH 7.4 was maintained by drop-wise addition of 0.1 N NaOH. After this period the pH remained stable. The extract was clarified by centrifugation and the supernate was extracted v/v with petroleum ether (Fisher Scientific, Pittsburgh, PA). The aqueous phase of this extraction was further extracted v/v with ethyl ether (Fisher Scientific). The aqueous phase was retained and stirred in a beaker in a fume hood to remove ether vapors. The extract was then concentrated with pressure on an Amicon PM10 membrane (Amicon Corporation, Danvers, MA). Solute that did not pass the PM10 filter and that which did were retained separately. The latter was then concentrated by pressure dialysis on an Amicon YM1 membrane. The fraction that did not pass this filter and the fraction that did were both retained separately. In this way, solutes that were >10,000 molecular weight (MW), between 10,000 MW and 1,000 MW, and <1,000 MW were obtained. The material used in these studies was from the <1,000 MW fraction. It is referred to as CSC. It was sized further on Sephadex G10 (Sigma Chemical Co., St. Louis, MO) and determined to be <700 MW. The concentration of CSC in mg/ml was determined by freeze drying and weighing. CSC contains little protein as measured with the Bradford assay8 but is very reactive in a tannin assay9 and most likely the majority of the reagent is polyphenolic. An equimolar complex of rutin, chlorogenic acid, and scopoletin, the three major polyphenol components of tobacco,10 has been identified in cigarette smoke and CSC may be related to this complex.11 Before use, CSC was filtered through Detoxi-Gel (Pierce Biochemicals, Rockford, IL), to remove any endotoxin in the preparation. This step was critical because others have shown that lipopolysaccharide (LPS) in the presence of human serum as a source of LPS-binding protein will prime neutrophils.12 As an additional precaution the studies reported here were performed in the absence of human serum. All of the media and buffers were purchased as endotoxin-free reagents.
The amount of tobacco-associated phenolics that a smoker inhales has been shown to range from 101 to 185 µg/cigarette.13,14 Smokers who smoke two packs a day could easily inhale up to 7,200 µg of polyphenols per day. Because these substances are of low molecular weight (300 to 700 daltons) and would be expected to cross the pulmonary epithelium easily, the concentration of these substances in pulmonary capillary blood could well be in the range of the concentration used in these experiments. Studies on the pharmacokinetics of the polyphenics in tobacco smoke after inhalation have not been reported in the scientific literature.
Preparation of TAIA
Tobacco glycoprotein (TGP) was extracted from tobacco leaves as described.15-17 TGP is a complex of tobacco leaf proteins and tobacco-associated polyphenols. Antibodies to TGP were generated in rabbits.18 The IgG fraction of the rabbit anti-TGP was isolated by affinity chromatography on immobilized Staphylococcal Protein A (Pierce Biochemicals). It was further purified by affinity chromatography and eluted from columns of TGP coupled to aminoalkyl-Sepharose columns with EDAC (Bio-Rad, Hercules, NY). BALB/c mice were immunized with the IgG fraction of rabbit anti-TGP in complete Freund’s adjuvant. The monoclonal anti-idiotypic antibody was prepared by fusing myeloma cell line P3x63Ag8.653 (ATCC CRL1580) with spleen cells from these BALB/c mice. Anti-idiotypic antibodies were identified by enzyme-linked immunosorbent assay using purified rabbit anti-TGP IgG, rabbit anti-rutin IgG (prepared as described previously18 ), rabbit anti-bovine serum albumin, and pooled rabbit IgG as target antigens. Confirmation of the anti-idiotypic specificity of the TAIA was established by demonstrating that monoclonal antibodies reactive with rabbit anti-TGP IgG or rabbit anti-rutin IgG but not rabbit anti-bovine serum albumin IgG or normal rabbit IgG, could be competitively inhibited from binding to these antigens in the presence of TGP, rutin, or chlorogenic acid, polyphenols found in tobacco leaves. The clone that produced the monoclonal antibody with the TAIA specificity was subcloned and an antibody of IgG1 subclass was isolated by chromatography on Bakerbond ABx columns (J. P. Baker Chemical Co., Phillipsburg, NJ). It is referred to as TAIA. Another mouse monoclonal of IgG1 subclass that reacts with an IgG myeloma protein and IgG from pooled mouse serum was also isolated on Bakerbond ABx columns. They are referred to as anti-id control and normal mouse IgG control, respectively. Before use, all antibody preparations were filtered through Detoxi-Gel (Pierce Biochemicals), to remove any endotoxin in the preparation. Protein concentrations were determined using the Lowry assay.19
Isolation of Neutrophils from Peripheral Blood
Peripheral blood was collected in sodium heparin (14.3 usp units/ml) from nonsmoking laboratory personnel. The neutrophils were isolated immediately after the blood draw. Blood (3.0 ml) was layered over 4.0 ml of Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) in a 12-ml polystyrene tube. The blood was allowed to settle at room temperature for 1 hour at which time the upper plasma/cell layer was removed. The plasma/cell mixture was then layered over 3.0 ml of Ficoll-Paque in a 12-ml tube and centrifuged at 1,000 x g for 15 minutes at 18°C. The neutrophils at the bottom of the tube were washed three times at 4°C in Hanks’ balanced salt solution (HBSS) (Life Technologies, Inc., Grand Island, NY) at 4°C. Residual red cells were lysed with hypotonic saline, neutrophils were washed again in HBSS and resuspended in the appropriate buffer. All experiments were completed within 3 hours of neutrophil isolation.
Flow Cytometric Analysis of CD11b/18
Anti-CD11b labeled with phycoerythrin, a marker for expression of CD11b/18, was purchased from Becton Dickinson (Mountain View, CA). Neutrophils were suspended to 5 x 106 cells/ml in PBS containing 5.0% glucose. A 0.2-ml aliquot of cells was mixed with 9 µg/ml of CSC or 10 µg/ml of TAIA or 10 µg/ml normal mouse IgG or PBS and incubated for 10 minutes at 37°C in a shaking water bath. Samples were cooled and received 20 µl of antiserum and 0.4 ml of ice-cold PBS and kept on ice for 10 minutes. To measure the effect of CSC on expression of CD11b/18 in whole blood, 9 µg/ml CSC or PBS were mixed with 0.2 ml whole heparinized blood and incubated for 10 minutes at 37°C. Each sample received 20 µl of antiserum and was placed on ice for 10 minutes. A Coulter Q Prep system (Coulter Corporation, Miami Lakes, FL) was used for lysing and fixing the stained cells. Analysis was performed using a Coulter EPICS Profile II. Results were expressed as a fold increase in CD11b/18 expression defined as the ratio of mean fluorescence intensity of cells incubated with CSC or TAIA and the mean fluorescence intensity of control cells incubated with PBS or control antibody.
Quantitation of fMLP Receptors
fMLP receptors on neutrophils were quantitated using an established assay.20 [3H]fMLP was purchased from New England Nuclear Life Sciences (Cambridge, MA). Aliquots (0.1 ml) of neutrophils at 1 x 106 cells/ml in HBSS, 1% heat-inactivated human AB+ serum and 5 mmol/L glucose were added to wells of microtiter plates. The cells were incubated with CSC at a final concentration of 9 µg/ml, 10 µg/ml TAIA, 10 µg/ml anti-id control, or buffer at 37°C. Varying volumes of diluted [3H]fMLP were added for final concentrations of 10 to 300 nmol/L. After 1 hour at 37°C the cells were collected on Whatman GF/B glass fiber filters using a PHD Cell Harvester (Cambridge Technology, Watertown, MA). The filters were counted using a Packard 2000CA Liquid Scintillation Counter (Meridan, CN). Scatchard plot analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA) and the number of fMLP receptors per cell was calculated.
Quantitation of Albumin Released in Response to CSC
Neutrophils isolated from peripheral blood were washed five times in PBS and suspended to 5 x 106 cells/ml in PBS. Cells were incubated at 37°C in a shaking water bath with either CSC (9 µg/ml) or PBS for 0, 5, and 10 minutes. The cells were centrifuged, the supernate was collected and analyzed for human albumin in a sandwich enzyme-linked immunosorbent assay. Ninety-six well enzyme-linked immunosorbent assay plates were coated with rabbit anti-human albumin (DAKO, Carpinteria, CA) at a final concentration of 5 µg/well. SuperBlock (Pierce Biochemicals) diluted in PBS was used as the blocking buffer. A standard curve was included in each assay using human albumin from ICN HSA Biochemicals (Costa Mesa, CA). The assay was developed using rabbit anti-human albumin horseradish-peroxidase-conjugated antibody (DAKO) and O-phenylenediamine (Sigma Chemical Co.) as the substrate. The standard curve was generated using StatView (Abacus Concepts, Inc., Berkeley, CA)
Quantitation of Neutrophil Superoxide Production
An assay to quantitate superoxide produced by neutrophils after stimulation with fMLP was performed as described.21,22 Purified neutrophils at 6 x 105 cells/ml in HBSS were incubated for 10 minutes at 37°C with either 9 µg/ml of CSC, 10 µg/ml of TAIA, or 10 µg/ml antibody control or PBS. Superoxide dismutase (Sigma Chemical Co.) at a final concentration of 750 U/ml or buffer and cytochrome c were added. Cells were stimulated with 1 µmol/L fMLP in a final volume of 1 ml for 1 hour at 37°C. After centrifugation, the supernatant was collected and read on a UV/VIS Spectrometer Lambda 10 (Perkin-Elmer, Norwalk, CT). The amount of superoxide anion produced per neutrophil was calculated from the amount of cytochrome c reduction inhibited by superoxide dismutase.
Quantitation of Neutrophil Elastase
Aliquots of 5 x 105
cells received CSC
at a final concentration of 9 µg/ml, TAIA at 10 µg/ml, antibody
control at 10 µg/ml or PBS. After incubation for 10 minutes at
37°C, fMLP was added to each tube at a final concentration of 1
µmol/L and incubation continued for 45 minutes with shaking. The
tubes were centrifuged and the elastase activity in the cell supernate
was measured.
L-Pyroglutamyl-S-prolyl-L-valine-p-nitroaniline
(S2484; Chromogenix, Molndal, Sweden), a synthetic substrate that is
specific at pH 8.3 for neutrophil elastase was used in these studies.
The assay for measuring elastase using the synthetic substrate was
performed at 37°C according to published procedures.23
In preliminary experiments performed to determine whether CSC
interfered with the synthetic substrate assay for elastase, neutrophil
elastase was prepared as described24
and allowed to cleave
S2484 in the presence or absence of CSC. CSC was found to not interfere
with the enzymatic activity of elastase as measured in this assay.
International units/ml (IU/ml) of elastase were calculated using
O.D./minutes and an absorption coefficient for
p-nitroaniline of 9.9 cm2/mmol/L at
405 nm.
Statistical Analysis
The number of times each experiment was performed is included with the results for the experiment. Statistical differences (t-test), means, and SEM were calculated using StatView (Abacus Concepts, Inc.). In these studies, the threshold P value for statistical significance is P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils were mixed with CSC, TAIA, anti-id control, or PBS and
incubated for 10 minutes at 37°C in a shaking water bath, cooled on
ice, stained with phycoerythrin-labeled anti-CD11b, and analyzed by
flow cytometry. The results are shown in Figure 1
. After incubation with CSC or TAIA,
CD11b/18 expression on the neutrophils more than doubled
(P < 0.01). To rule out the effect of CSC on
expression of CD11b/18 on neutrophils being an artifact associated with
the use of purified cells, CSC was added to whole blood collected in
heparin. Also shown in Figure 1
, after 10 minutes at 37°C there was a
threefold increase (P < 0.01) in expression of
CD11b/18 on neutrophils in whole blood. fMLP receptor expression was
also measured on purified neutrophils primed with CSC and TAIA. As
shown in Figure 2
, after incubation with
CSC or TAIA, the number of fMLP receptors on the neutrophils doubled
compared to the controls (P < 0.05).
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CD11b/18, fMLP receptors, alkaline phosphatase, cytochrome
b558, albumin, and other plasma proteins are
found in secretory vesicles, the most readily mobilized compartment in
neutrophils.25
Our data suggest that one effect of CSC and
TAIA on neutrophils is to mobilize these vesicles to the plasma
membrane resulting in translocation of CD11b/18 and fMLP receptors to
the cell surface. As part of this process, the albumin contained within
the secretory vesicles is released into the cell
supernate.25
Neutrophils isolated from peripheral blood
were washed extensively to remove serum proteins bound to the cell
membrane. Baseline albumin concentrations were achieved by adding PBS
or CSC to neutrophils at 4°C, followed by immediate centrifugation.
As shown in Figure 3
, there is some
release of albumin from cells incubated with PBS. This is not
unexpected because a slight increase in expression of CD11b/18 occurs
normally when purified cells are incubated at 37°C.26
In
the presence of CSC, however, after 5 minutes there was a 1.6-fold
increase in albumin in the supernate compared with cells incubated with
PBS. By 10 minutes, 2.5-fold more albumin had been released from the
CSC-treated cells. These results provide evidence that one effect of
CSC and TAIA is to mobilize secretory vesicles thereby increasing
CD11b/18 and fMLP receptor expression on the primed cells.
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Production of superoxide by neutrophils primed with CSC or TAIA
before stimulation with fMLP was quantitated using a superoxide
dismutase-inhibitable cytochrome c assay. As shown in Table 1
, the neutrophils primed with CSC
produced 50% more superoxide anion per cell which was a significant
increase (P < 0.003) over the response of
unprimed cells. Cells primed with TAIA produced 20% more superoxide
than cells preincubated with a control IgG (P <
0.003).
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Neutrophil elastase has been implicated as a major contributor to
the inflammatory process especially in inflammatory lung diseases like
COPD where it functions to destroy the architecture of the alveolar
walls. Neutrophils were primed with CSC or TAIA and release of elastase
after addition of fMLP was quantitated. As shown in Table 2
, there was a 1.94-fold increase over
control (P < 0.001) in elastase release when
neutrophils were primed with CSC. Neutrophils primed with TAIA showed a
2.09-fold increase (P < 0.001) in elastase
release. In these experiments, neutrophils were incubated with the
priming agent for 10 minutes followed by 45 minutes of incubation with
fMLP or PBS. There was some elastase released from the cells in the
absence of fMLP but the difference between spontaneous release of
elastase in the presence of CSC or PBS was not significant (Table 2)
.
This can be interpreted to mean that CSC alone is not functioning as an
activating agent. Similarly, even though spontaneous release of
elastase in the presence of mouse IgG control or TAIA was increased
compared with CSC or PBS the response to the two mouse immunoglobulin
reagents was not significantly different.
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A role for MAPK p38 activation and phosphorylation in neutrophil
priming by CSC and TAIA was examined using SB203580 (Calbiochem), a
specific p38 inhibitor.27,28
Neutrophils at
107
cells/ml were mixed with buffer or SB203580
at a final concentration of 500 nmol/L and incubated for 10 minutes at
37°C. Aliquots (0.2 ml) of inhibitor-treated or -untreated cells
received CSC at 9 µg/ml or PBS. After 10 minutes at 37°C, the cells
were cooled on ice, stained with phycoerythrin-labeled anti-CD11b, and
analyzed by flow cytometry. As shown in Figure 4
, CSC caused a twofold or 100% increase
in CD11b/18 expression (P < 0.01). With the
cells that had been pretreated with SB203580, the increase in
expression of CD11b/18 after incubation with CSC was only 20% and did
not differ significantly from the PBS control.
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and show a significant
(P < 0.03) increase in superoxide production,
as would be expected, when neutrophils were primed with CSC. When the
cells were pretreated with SB203580 before CSC their response to fMLP
was similar to the response of unprimed cells suggesting SB203580 was
preventing the priming process from occurring. The observation that
SB203580 treatment alone did not significantly reduce the ability of
the neutrophils to produce superoxide in response to fMLP further
supports its role as an agent that interferes with the priming
mechanism. The results suggest that blocking activation and
phosphorylation of MAPK p38 with a specific inhibitor prevents
neutrophil priming by CSC. In these experiments, the effect of SB203580
was to abrogate priming by CSC and it did not significantly affect the
ability of the cells to respond to fMLP activation as measured by
superoxide production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the concept of tobacco anti-idiotypic
antibodies mimicking CSC to prime neutrophils is diagrammatically
illustrated.
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, interleukin (IL)-8, LPS, and granulocyte macrophage-colony
stimulating factor,29
have demonstrated that an increase
in fMLP receptors is not necessarily required for an increased
oxidative burst response to fMLP activation. Zimmerli et
al30
using neutrophils primed with LPS found neutrophil
oxidative burst was increased in response to C5a in the absence of
up-regulation of C5a receptors. These results argue against there being
a direct association between an increase in receptor number and the
responsiveness of primed cells. Studies on the response of CSC- and
TAIA-primed cells to agents other than fMLP with receptors that are not
so easily mobilized are currently underway. Up-regulation and activation of CD11b/18 on cigarette smoke-primed neutrophils may be very important for recruiting and maintaining these cells at inflammatory foci. A result of up-regulation and activation of CD11b/18 coupled with an increase in ICAM-1 in response to infection or injury would be increased leukocyte adhesion, emigration, and tissue damage in smokers. We used the increase in expression of CD11b/18 in response to CSC as a marker of priming and showed that it could be prevented using SB203580, an inhibitor specific for MAPK p38 activation and phosphorylation. Further, enhancement of the oxidative burst of neutrophils primed with CSC and challenged with fMLP was abrogated in the presence of SB203580. It is not surprising that CSC, a plant-derived product would use MAPK pathways. Other polyphenols extracted from green tea have been shown to activate ERK and JNK MAPK in human hepatoma HepG2 cells.31 In plants, MAP kinases represent an ancient signaling mechanism, are implicated in a wide variety of biological responses, and play a role in signaling of abiotic stress, pathogens, and plant hormones.32
The premise for our work is that in smokers, primed neutrophils
contribute to the occurrence and progression of cigarette
smoking-associated diseases that are inflammatory in origin. After
cessation of smoking, the presence of tobacco anti-idiotypic antibodies
functionally similar to priming agents in cigarette smoke may continue
to drive the process. A variety of anti-idiotypic antibodies have been
described in human serum. For example, pooled normal human IgG prepared
for therapeutic use has been shown to contain antibodies reactive with
anti-microsomal antibodies from patients with Graves disease,
postpartum thyroiditis, and autoimmune thyroiditis through
idiotypic-anti-idiotypic interactions.33
As part of the
immune network, the natural occurrence of anti-idiotypic antibodies may
be extremely common. Generation of tobacco anti-idiotypic antibodies
would require an initial immune response to tobacco antigens. When
challenged intradermally with a TGP preparation, at least one third of
smokers and nonsmokers tested developed an immediate, cutaneous
hypersensitivity wheal and flare reaction, indicating an IgE-mediated
response.34
This suggests that an immune response to
tobacco is not a rare event. Constituents of cigarette smoke may help
drive the immune system by stimulating release of IL-1, IL-1ß,
IL-6, and PDGF from macrophages.35
They also cause release
of IL-4 from TH2 lymphocytes which drives B-cell
immunoglobulin production.36,37
As a general rule,
removing the antigen should result in eventual clearance of antibodies
to tobacco with consequent reduction in production of anti-idiotypic
antibodies. Even after smoking has stopped, this network balancing may
not take place because there will be continued stimulation of the
immune system by smaller amounts of antigen in side-stream
smoke38
coupled with cross-reactive antigens from other
environmental sources.18
Other examples of anti-idiotypic antibodies functioning like the original antigen include antibodies with insulin-like activity,39 antibodies that mimic tumor antigens, and an anti-idiotypic antibody that mimics the functional activity of Paclitaxel (Taxol) a low molecular weight anti-tumor agent.40 The Paclitaxel anti-idiotypic antibody model is an example especially relevant to our work because both Paclitaxel and tobacco polyphenols share aromatic ring structure and bear no resemblance to immunoglobulin molecules. Yet both the Paclitaxel-like anti-idiotypic antibody and TAIA display functional activity identical to the original antigen.
In the next phase of our studies we will quantitate tobacco anti-idiotypic antibodies in serum from healthy smokers and smokers with smoking-associated disorders like COPD. In preliminary studies, six of 11 (55%) patients with clinically documented COPD (all previous or current smokers) and one of nine (11%) serum samples from disease free, long-term smokers (up to 45 years smoking history) had TAIAs as measured in an enzyme-linked immunosorbent assay. This study will be expanded to include more patients, TAIA will be isolated by affinity chromatography and the functional activity of the TAIA as a neutrophil-priming agent will be measured. The hypothesis is that some smokers produce TAIA functionally similar to CSC that drive progression and even aid in initiation of cigarette smoking-associated inflammatory diseases long after smoking has stopped. This hypothesis is novel but the in vitro results provide evidence that the postulate it is based on is sound. It is now critical to demonstrate antibodies to tobacco antigens and anti-idiotypic antibodies in patients and correlate these findings with course and severity of smoking-associated diseases.
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Footnotes |
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Supported by a grant from the American Lung Association of Wisconsin.
Accepted for publication July 20, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 antitrypsin deficiency. Am Rev Respir Dis 1980, 121:253-261[Medline]
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) compared with cells
incubated in PBS (•).
