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Rapid lung cytokine accumulation and neutrophil recruitment after lipopolysaccharide inhalation by cigarette smokers and nonsmokers
Rapid lung cytokine accumulation and neutrophil recruitment after lipopolysaccharide inhalation by cigarette smokers and nonsmokers LEWIS J. WESSELIUS, MICHAEL E. NELSON, KIRSTIN BAILEY, and AMY R. O'BRIEN-LADNER KANSAS CITY, MISSOURI
Inhalation of lipopolysaccharide (LPS) by humans rapidly recruits neutrophils to alveolar structures. Recruitment of neutrophils may be mediated in part by intrapulmonary release of cytokines such as tumor necrosis factor-s, interleukin (IL)- 113,and IL-8, although the kinetics of cytokine accumulation and neutrophil recruitment to the lungs after LPS inhalation have not been determined. Release of some cytokines in response to LPS is reported to be decreased in smokers' alveolar macrophages compared with nonsmokers', suggesting responses to LPS may differ in smokers (S) and nonsmokers (NS). To assess the kinetics of early cytokine accumulation after LPS inhalation and to compare inflammation induced in LPS-exposed S and NS, we performed bronchoalveolar lavage (BAL) in 28 subjects (14 NS and 14 S) at 90 or 240 minutes after inhalation of aerosolized LPS (30 ~g). BAL performed at 90 and 240 minutes after LPS inhalation recovered increased numbers of neutrophils and lymphocytes in both NS and S compared with an unexposed control group (10 NS, 10 S), with greater recovery of neutrophils in S than NS (p < 0.00 I). BAL fluid supemate concentrations of IL-8, IL-113, and tumor necrosis factor-oL at 90 minutes were increased in S and NS compared with an unexposed control group. IL-8 and tumor necrosis factor-~ concentrations were similar in S and NS; however, IL- 113 concentrations were greater in S (p < 0.005). BAL fluid concentrations of IL-113 and IL-8 at 90 minutes correlated with absolute neutrophil recovery in S and NS. These findings suggest that the rapid accumulation of cytokines, particularly IL-113 and IL-8, contributes to lung neutrophil recruitment after LPS inhalation. In addition, parameters of pulmonary inflammation present in S after LPS inhalation are similar to or increased compared with those present in NS. (J Lab Clin Med 1997;129:106-14)
Abbreviations: BAL = bronchoalveolar lavage; ELISA = enzyme-linked immunosorbent assay; IL-8 = interleukin-8; IL-113= interleukin-1 beta; LPS = lipopolysaccharide; TNF-~ = tumor necrosis factor alpha
From the Divisionof Pulmonaryand Critical Care Medicine, Department of Medicine, University of Kansas Medical Center, and Department of Medicine, Department of Veterans AffairsMedical Center. Supported by American Heart Association, Kansas Affiliate, and by the Research Service, Department of Veterans Affairs. Submitted for publication July 15, 1996; revision submitted July 15, 1996; accepted Aug. 1, 1996. Reprint requests: Lewis J. Wesselius, MD, Department of Medicine (111), Department of Veterans Affairs Medical CenterKansas City, 4801 LinwoodBlvd., Kansas City, MO 64128. Copyright © 1997 by Mosby-Year Book, Inc. 0022-2143/97 $5.00 + 0 5/1/77480 106
here is considerable evidence that LPS present in organic dusts is an important mediator of pulmonary inflammation associated with exposure to grain, cotton, and swine dusts. 1'2 Inhalation of LPS by human subjects induces an intense inflammatory response characterized by rapid neutrophil and lymphocyte recruitment to lung structures. 3'4 Inhalation of LPS in sufficiently high doses can induce airway dysfunction, and longer term exposure to smaller amounts of inhaled LPS may cause chronic cough and increased sputum production and may lead to accelerated loss of lung function. 5-8
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Previous studies indicate that intrapulmonary instillation of LPS in rats induces the expression of inflammatory cytokines by alveolar macrophages. 9 Inhalation of LPS-containing grain dusts by h u m a n subjects also has b e e n shown to induce expression of IL-8, TNF-c~, and IL-113 by alveolar macrophages. 1° The intrapulmonary accumulation of macrophagederived cytokines such as IL-8 and IL-113 may contribute to early neutrophil recruitment after LPS inhalation in humans, 11-14 although cytokines may also be derived from other cells such as neutrophils, epithelial cells, or fibroblasts, ls-17 Neutrophil recruitment to the lungs occurs within 3 hours of LPS inhalation by h u m a n volunteers, suggesting that cytokine accumulation must occur rapidly to mediate neutrophil recruitment. The early time course of cytokine and neutrophil accumulation within h u m a n lungs after LPS inhalation has not previousb, been determined. Alveolar macrophages recovered from cigarette smokers are reported to release decreased amounts of inflammatory cytokines including I L - I ~ and TNF-oL after in vitro exposure to LPS compared with alveolar macrophages recovered from nonsmokers. ls-2° This observation suggests that the pulmonary inflammatory response to LPS inhalation in smokers may be diminished compared with that in nonsmokers. Clinical observations, however, suggest t h a t cigarette smoking is associated with enhanced risk of pulmonary dysfunction in response to chronic exposures to inhaled L P S Y '22 In this study we compared pulmonary inflammation associated with LPS inhalation in a group of healthy smokers with a group of never-smokers to determine whether there w a s evidence of diminished lung inflammatory responses to inhaled LPS in smokers. METHODS Study subjects and spirometry. The LPS-treated study population consisted of 28 subjects including 14 nonsmokers and 14 current smokers studied at either 90 minutes (7 nonsmokers, 7 smokers) or 240 minutes (7 nonsmokers, 7 smokers) after inhalation of LPS. Smokers had a minimum smoking history of 10 pack years. In addition, we studied 20 matched subjects (10 nonsmokers and 10 smokers) who were not treated with LPS and who served as control groups. No subject in the study was taking medications or reported taking aspirin or nonsteroidal medications within the previous 2 weeks. All subjects denied chronic production of sputum, and none reported a recent history of upper respiratory tract infection. All subjects had baseline spirometry performed with a pneumotachograph (Medical Graphics System 1070, St Paul, Minn.), and the best of three efforts was recorded. All subjects studied 4 hours after LPS inhalation had spirometry re-
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peated immediately before bronchoscopy. The protocol used was approved by the Human Subjects Committee of the Kansas City Department of Veterans Affairs Medical Center. Written informed consent was obtained from all subjects. LPS inhalation. A solution of LPS was made by adding LPS (Escherichia coli, serotype 0111:B4, Sigma, St. Louis, Me.) to sterile normal saline solution. A solution containing 30 mg LPS in 3 ml saline solution was inhaled by subjects over approximately 15 minutes with a nebulizer (Respirgard II, Marquest, Englewood, Colo.) and a Pulmo-Aid machine. This dose of inhaled LPS was chosen on the basis of preliminary studies demonstrating neutrophil recruitment in the absence of a significant reduction in FEV 1 and is consistent with previous studies. 5 Bronchoscopy and bronchoalveolar lavage. Subjects received light sedation with intravenous meperidine. After the oropharynx was anesthetized with tetracaine, brenchoscopy was performed by the oral route. Bronchoalveolar lavage was performed in subsegments of the middle lobe and lingula; 6 aliquots of 20 ml of sterile 0.9% saline solution were instilled for a total of 120 ml lavage in each lung. Lung lavage fluid was recovered by gentle suction and was sequentially processed with the initial 20 ml aliquot (bronchial sample) collected separately from the following 4 aliquots (alveolar aliquots), which were pooled for analysis. Processing of specimens. BAL fluid was immediately filtered over sterile gauze to remove mucous. Cells were recovered by centrifugation (400g), and aliquots of BAL fluid were decanted and stored at - 7 0 ° C until subsequent analysis was performed. Cell recovery was determined with a hemacytometer, and a cell differential was determined by counting 200 cells on a Giemsa/Wright-stained cytocentrifuge preparation. Cytokine and protein determinations. The concentrations of IL-8, TNF-c~, and IL-113 present in bronchoalveolar lavage supernates and serum obtained at the time of bronchoscopy were determined with a standard ELISA (R&D Systems, Minneapolis, Minn.) with antibodies raised against human recombinant IL-8, TNF-a, and ILl[3. The sensitivity of the ELISA assays used is as follows: IL-8 assay is sensitive to 3 pg/ml, the TNF-c~ assay is sensitive to 4.4 pg/ml, and the IL-l[3 assay is sensitive to 0.3 pg/ml. We ran all samples in duplicate. The ELISA assays we used are not reported to exhibit detectable cross-reactivity with the other cytokines we tested. Protein content of BAL fluid was determined with a bicinchoninic acid protein assay reagent (BCA kit; Pierce, Rockford, Ill.) with bovine serum albumin used as a standard. Statistical analysis. Data are expressed as mean +_ SE. Differences between groups were analyzed with the Mann-Whitney rank sum test. In all tests statistical significance was identified at the p < 0.05 level. We conducted least squares linear regression analysis to examine the relationships between BAL fluid supernate content of IL-8 and IL-113 after LPS inhalation and numbers of neutrophits in recovered cells.
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Table I. C h a r a c t e r i z a t i o n
of study populations
Smoking history (pack yeors)
FEV1% pred/ FVC % pred
36.3 _+ 2.2
0
38.1 _+ 1.6
27 _+ 3
102 _+2 104 _+ 2 92_+4 104 -+ 2
LPS-Exposed 90 minutes Nonsmokers (n = 7)
36.4 _+ 2.1
0
Smokers (n = 7)
40.8 _+ 2.3
34 -+ 4
34.0 _+ 2.4
0
38.7 + 2.7
31 _+ 4
Age (yr) Control group Nonsmokers (n = 10) Smokers (n = 10)
240 minutes Nonsmokers (n = 7) Smokers (n = 7)
98_+3 99_+2 88 _+4 101 -+2 101 103 91 102
-+3 _+ 3 _+4 __ 3
Values expressed as mean _+SE.
Table II. C e l l a n d p r o t e i n r e c o v e r y in BAL f l u i d Total cells (× 106) No LPS Nonsmokers Smokers After LPS 90 minutes Nonsmokers Smokers 240 minutes Nonsmokers Smokers
Alveolar macrophage (× 10n/ml)
Neutrophils (x 104/mi)
Lymphocytes (x 104/mi)
Protein (mg/ml)
11.7 + 0.8 26.4* -+ 3.0
7.9 -+ 0.5 19.1" _+ 3.1
0.03 _+ 0.01 0.24 + 0.06
0.38 -+ 0.10 0.44 -+ 0.15
.088 -+ .010 .119 -+ .019
13.3 -+ 1.1 30.3* -+ 3.7
8.5 -+ 0,4 25,5" + 3.7
1.621- -+ 0,6 4.51" _+ 1.28
0.47 + 0.15 0.61 _+ 0.13
.117 -+ .018 .138 -+ .019
17.3 + 2.4 47.2* + 13.9
11,11- + 1.3 31.8" -+ 7.0
2.131- -+ 0.52 7.05* _+ 2.86
0.811- _+ 0.18 0.961- + 0.20
.127 -+ .010 .143 + .021
*p < 0.05 compared with nonsmokers. 1-P < 0,05 compared with no LPS.
RESULTS Subject characteristics and spirometry. The charac-
teristics of the study populations are provided in Table I. All of the smoking subjects were current smokers and had at least a 10 pack year total smoking history (range 10 to 50 pack years). Smoking histories and ages were similar in the control group (not LPS-exposed) and in subjects studied 90 or 240 minutes after LPS inhalation. Mean baseline spirometric measurements for each group are also provided in Table I. The mean FEV1 was lower in smoking subjects than in nonsmokers; however, all smokers had an FEV1 that was greater than 70% of the predicted value. Spirometry performed just before bronchoscopy in all subjects studied 4 hours after LPS inhalation demonstrated that
none of the subjects had a significant (>10%) decrease in FEV1 compared with baseline (data not shown). BAL fluid cellularity. Bronchoalveolar lavage performed in subjects without exposure to LPS recovered significantly more cells in smokers than in nonsmokers primarily as a result of increased recovery of alveolar macrophages (Table II). Subjects studied after LPS inhalation had an increase in total cell recovery at both 90 and 240 minutes, which was a result of increased recovery of both neutrophils and alveolar macrophages. Significantly greater numbers of neutrophils were recovered by BAL in smokers at both 90 and 240 minutes after LPS than in nonsmokers (Table II). Also, significantly greater numbers of lymphocytes were recovered by bronchoalveolar la-
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2800-
2400-
2000-
IL-8 pg/ml 1600-
1200-
OO
800-
400-
_~.
o o0
%.
NS S p<.05 NO LPS
NS
S
NS I
n.s. 90 min
I S I
n.s.
240 min TIME AFTER LPS
Fig. 1. Concentrations of IL-8 in B A L tluid supernates recovered from groups either exposed to no LPS (control) or groups studied at 90 or 240 minutes after inhalation of LPS (30 rag). * = p < 0.05 compared with concentrations at 90 minutes, n.s. = not significantly different.
vage in both smokers and nonsmokers at 240 minutes after inhalation of LPS with similar increases in both groups (Table II). BAL fluid concentrations of cytokines and protein.
Evaluation of BAL fluid supernatants recovered from subjects without exposure to LPS demonstrated a small but significant increase in IL-8 concentrations in smokers compared with nonsmokers (p < 0.05) (Fig. 1). In several smokers BAL fluid supernatant concentrations of IL-I[3 were also greater than values present in nonsmokers; however, differences between groups were not significant (Fig. 2). BAL fluid supernatant concentrations of TNF-~ were similar in both smokers and nonsmokers (Fig. 3). The total protein content of BAL fluid was slightly higher in smokers than in nonsmokers as has been noted in previous studies.23 Concentrations of IL-8, TNF-oL, and IL-113 in recovered BAL fluid were increased at 90 and 240 minutes after LPS inhalation in both nonsmokers
and smokers compared with respective unexposed groups (Figs. 1, 2, and 3). Concentrations of IL-8 and TNF-a were not significantly different in smokers studied 90 or 240 minutes after LPS inhalation compared with nonsmokers. In contrast, BAL fluid concentrations of IL-I[3 were significantly increased in smokers compared with nonsmokers in subjects studied both at 90 and 240 minutes after LPS (Fig. 3). Concentrations of TNF-oL and IL-113 in BAL fluid supernates were not significantly different at 240 minutes compared with 90 minutes after LPS inhalation in either smokers or nonsmokers. In contrast, concentrations of IL-8 were significantly lower at 240 minutes compared with 90 minutes (0 < 0.05) in both smokers and nonsmokers. Protein concentrations in BAL fluid after LPS inhalation were increased in both smokers and nonsmokers compared with the unexposed control group at 90 minutes; however, differences were not significant (Table II). A small but significant in-
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120 O0
100
0
O0
e
TNF pg/ml
80
eeee
0
o
O0 0
60'
O0
0
ee
40-
0
o
ee
20-
•
oooo
ee
oo
m=aa
I
nnn~
NS '
S
I
I
n.s.
NO LPS
NS
I
I
S I
NS I
I
n.s,
n.s.
90 min
I S
240 min TIME AFTER LPS
]Fig. 2. Concentrations of TNF-a in BAL fluid supernates recovered from groups either exposed to no LPS (control) or groups studied at 90 or 240 minutes after inhalation of LPS (30 mg). n.s. = not significantly different.
crease occurred in total protein content of BAL fluid at 240 minutes after LPS inhalation in both smokers and nonsmokers (Table II). No difference was seen in the absolute change in BAL fluid protein content in smokers or nonsmokers. Serum cytokine concentrations. S e r u m c o n c e n t r a -
of TNF-oL and IL-113 were determined in unexposed subjects and 90 or 240 minutes after LPS inhalation in exposed subjects. In unexposed subjects serum concentrations of TNF-oLwere undetectable and serum concentrations of IL-I[3 were low or undetectable. At 90 and 240 minutes after LPS inhalation serum TNF-oL was detectable in 23 of 28 subjects, and values were similarly increased in smokers and nonsmokers (Table III). Serum concentrations of IL-113were similarly increased in both groups at 90 and 240 minutes after LPS inhalation compared with unexposed subjects (Table III). Correlations. To assess whether there was a relationship between IL-113 concentrations in BAL fluid and lung neutrophil recruitment, we evaluated for correlations between these values in smokers and nonsmokers. Statistical analysis demonstrated a cortions
relation between IL-l[3 concentrations in BAL fluid supernates recovered at 90 minutes after LPS inhalation and the numbers of neutrophils in cells recovered by BAL (Fig. 4). The correlation between IL-113 concentrations and absolute neutrophil recovery (r = .71, p < 0.05) was slightly greater than the correlation between IL-8 concentrations and absolute neutrophil recovery (r = 0.64, p < 0.05). DISCUSSION
The data obtained in this study indicate that there are marked increases in alveolar concentrations of IL-8, IL-I[3, and TNF-R in human subjects within 90 minutes of inhalation of LPS, supporting the concept that these cytokines contribute to early lung neutrophil recruitment induced by LPS. In addition, in this study we studied both smokers and nonsmokers after LPS inhalation and found that both groups had similar BAL fluid concentrations of IL-8 and TNF-~, but smokers had increased concentrations of IL-I[3 compared with nonsmokers. Recovery of neutrophils by BAL after LPS inhalation was also greater in smokers than nonsmokers, indicating that
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160-
140-
120-
100-
1L- ll] pg/ml
80-
60-
O
40-
o
20o
•. "" NS
e
_~_
!
S
NS
I
I .... n.s.
I
..
I
S
I
NO LPS
NS
I
I
I
S 1
p<.005
p<.005
90 min
240 min TIME AFTER LPS
Fig. 3. Concentrations of IL-113 in BAL fluid supernates recovered from groups either exposed to no LPS (control) or groups studied at 90 or 240 minutes after inhalation of LPS (30 mg). n.s. = not significantly different.
Table III. TNF-= a n d IL-113 c o n c e n t r a t i o n s in serum ( p g / m l ) No LPS* TNF-c~
Nonsmokers Smokers
ND ND
90 Minutes I L- I I~
0.3 -+ 0.1 0.3 -+ 0.1
240 Minutest
TNF-c~
I L- 113
TNF-~
92:1: -+ 19 805 -+ 15
1.3:1: -+ 0.3 1.45 -+ 0.2
81:1: + 15 655 -+ 6
IL- 113
5.0:1: +- 0.2 3.1:1: -+ 0.4
*p < 0.05 compared with No LPS group. 1-n = 7 for 90 and 240 minute groups in nonsmokers and smol, ers. *n = 10 for No LPS group in nonsmokers and smokers. ND, Not detected in any subject.
pulmonary inflammation associated with LPS inhalation is not diminished in smokers compared with nonsmokers. In our studies of control (not LPS-exposed) smokers and nonsmokers we found evidence of pulmonary inflammation associated with smoking alone including increases in BAL fluid concentrations of IL-8 and increased recovery of neutrophils in smokers compared with nonsmokers, findings that are
consistent with previous studies, ls'24 Because we did not determine baseline neutrophil recovery or cytokine concentrations in BAL fluid for each LPSexposed subject, we cannot determine a percentage increase in these parameters for individual subjects and therefore cannot determine with certainty whether the effects of LPS inhalation are additive or synergistic with smoking exposures in the induction of inflammation. Concentrations of IL-I~ in BAL
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!60 140 120 100 L_
°
•
It-113
pg/ml
80 604020
J
O 9/0
p < .05
O • o
I
I
I
I
I
I
t
I
I
I
I
I
1
2
3
4
5
6
7
8
9
10
11
12
Neutrophil Recovery x104/ml
Fig. 4. Relationshipbetween concentrationof IL-113in BAL fluidsupernates and absolute numbers of neutrophilsin cellsrecoveredby BAL.Values are fromgroups studiedat 90 minutesafterLPS inhalation and includenonsmokers(open circles) and smokers (closed circles).
fluid of LPS-exposed nonsmokers were sixfold greater than in unexposed nonsmokers, whereas IL-l[3 concentrations were approximately twelvefold greater in LPS-exposed smokers than in unexposed smokers. This finding suggests the possibility that intrapulmonary accumulation of IL-113 after LPS inhalation may be enhanced in some smokers. Because our smoking population was slightly older than the nonsmoking population and had lower FEV 1 values, these variables could also have contributed to the differences we observed. The accumulation of proinflammatory cytokines in the lower respiratory tract after LPS exposure has been proposed to contribute to lung inflammation and injury associated with LPS exposures) ° Previous studies indicate that intrapulmonary administration of IL-8 or IL-I[3 in experimental animals induces substantial recruitment of neutrophils to the lungs, so the accumulation of both of these cytokines within the lungs by 90 minutes after LPS inhalation supports the concept that these cytokines contribute to early LPS-induced recruitment of neutrophils to the lungs./1'13 Because IL-8 is also chemotactic for
lymphocytes, increased lung content of this cytokine may contribute to the accumulation of lymphocytes after LPS inhalation, which we noted, and which is similar to findings of previous studies.3'4'25 The presence of increased lung concentrations of IL-l[3 within alveolar structures after LPS inhalation may contribute to greater neutrophil recruitment by inducing enhanced expression of adhesion molecules on lung endothelial and epithelial cells, which could promote neutrophil recruitment. 26'27 The relatively greater concentrations of IL-113 present within the lungs of smokers may contribute to greater absolute numbers of neutrophils recovered from LPS-exposed smokers compared with nonsmokers. It is possible that differences in other neutrophil chemoattractants such as leukotriene 134 or GRO alpha may also contribute to differences in neutrophil recruitment. 2s'29 In addition to elaborating neutrophil chemoattractants, alveolar macrophages also release a low-molecular weight factor that inhibits neutrophil chemoattractant responses. 3° Diminished release of this inhibitory factor by smokers' alveolar macrophages could also poten-
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tially contribute to increased lung neutrophil recruitment in smokers. The finding of increased lung concentrations of IL-113 in smokers compared with nonsmokers after LPS inhalation is somewhat surprising, because alveolar macrophages recovered from smokers release substantially less IL-I[3 in response to in vitro LPS exposure than alveolar macrophages from nonsmokersJ 9'2° It is possible, however, that the greater number of alveolar macrophages present in smokers leads to a greater respiratory tract burden of alveolar macrophage-derived IL-113 after LPS exposures. Alterr~atively, IL-I~ may be derived from other cells such as neutrophils, which can be a source of IL-I[3 after endotoxin exposures. 31 Previous studies have demonstrated that human alveolar macrophages stimulated with LPS in vitro do not release significant amounts of IL-I[~ during the initial 4 hours after stimulation. 14 Our finding of IL-I[~ accumulation by 90 minutes after LPS inhalation suggests that either alveolar macrophage elaboration of IL-I[3 occurs more rapidly in vivo or that IL-113 accumulating initially after LPS inhalation is derived from cells other than alveolar macrophages. Although LPS inhalation was associated with recruitment of neutrophils to lung structures and increased lung concentrations of inflammatory cytokines in both smokers and nonsmokers, there was only a limited effect on lung vascular permeability. This finding is similar to a previous report in which leukotriene B4-induced lung neutrophil recruitment in human lungs was not associated with increased lung vascular permeability. 32 The absence of significant increases in lung vascular permeability despite increases in lung content of TNF-c~ and IL-l[3 suggests that the lung epithelial surface in humans is relatively resistant to injury from these cytokines at least at the concentrations induced in these studies. Serum concentrations of TNF-a and IL-l[3 were increased by 90 minutes after LPS inhalation, indicating a systemic inflammatory response was induced by LPS inhalation. These findings extend previous observations that LPS inhalation increased serum concentrations of TNF-c~ at 60 minutes after exposure in asthmatics and are consistent with recent reports on the time course of increased serum TNF-a after inhalation of LPS-containing organic dusts. 33'34 Increases in serum TNF-o~ may contribute to lung recruitment of neutrophils after LPS inhalation by inducing expression of adhesion molecules on lung endothelial cells. 35 In summary, this study indicates that inhalation of LPS by human subjects induces rapid accumulation of IL-8, IL-I~, and TNF-cx within the lower respira-
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tory tract. The kinetics of accumulation of IL-113 and IL-8 within alveolar structures after LPS inhalation are consistent with a role for both cytokines in early LPS-induced lung neutrophil recruitment. It is interesting that the accumulation of IL-lJ3 occurs more rapidly than is required for in vitro elaboration of IL-I[~ by LPS-stimulated alveolar macrophages. Comparison of pulmonary inflammation induced by LPS inhalation in smokers and nonsmokers indicates that BAL in LPS-exposed smokers recovered greater numbers of neutrophils and that recovered BAL fluid had greater concentrations of IL-I[3 compared with nonsmokers. The higher BAL fluid concentrations of IL-113 present in BAL fluid recovered may be a result of either additive inflammatory" effects of smoking and LPS exposure or an interaction between smoking and LPS exposure with regard to accumulation of intrapulmonary IL-I[3. The authors thank Dr. James Williams, Jr. for helpful suggestions in this study, and LaTrisha Gaston for her assistance in the preparation of the manuscript.
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five-year change in lung function. Am J Respir Crit Care Med 1994;150:1250-5. Wesselius LJ, Nelson ME, Skikne BS. Increased release of ferritin and iron by iron-loaded alveolar macrophages in cigarette smokers. Am J Respir Crit Care Med 1994;150: 690-5. Hunninghake GW, Crystal RG. Cigarette smoking and lung destruction: accumulation of neutrophils in the lungs of cigarette smokers. Am Rev Respir Dis 1983;128:833-8. Baggiolini M, Dewald B, Moser B. Interleukin-8 and related chemotactic cytokines--CXC and CC chemokines. Adv Immunol 1994;55:97-179. Luscinskas FW, Cybulsky MI, Kiely JM, Peckins LS, Davis VM, Gimbrone MA. Cytokine-activated human endothelial monolayers support enhanced neutrophil transmigration via a mechanism involving both endothelial-leukocyte adhesion molecule -1 and intercellular adhesion molecule -1. J Immunol 1991;146:1617-25. Roche WR, Montefort S, Baker J, Holgate ST. Cell adhesion molecules and the bronchial epithelium. Am Rev Respir Dis 1993;148(Suppl):S79-82. Martin TR, Altman LC, Albert RK, Henderson WR. Leukotriene B4 production by the human alveolar macrophage: a potential mechanism for amplifyinginflammation in the lung. Am Rev Respir Dis 1984;129:106-11. Becker S, Quay J, Koren HS, Haskill JS. Constitutive and stimulated MCP-1, GROa, b, and g expression in human airway epithelium and bronchoalveolar macrophages. Am J Physiol 1994;266(LungCell Mol Physiol 10):L278-286. Sibille Y, Merrill WW, Naegel GP, Care SB, Cooper JAD, Reynolds HY. Human alveolar macrophages release a factor that inhibits phagocyte function. Am J Respir Cell Mol Biol 1989;1:407-16. Williams JH Jr, Patel SK, Hatakeyama D, Arian R, Guo K, Hickey TJ, et al. Activated pulmonary vascular neutrophils as early mediators of endotoxin-induced lung inflammation. Am J Respir Cell Mol Biol 1993;8:134-44. Martin TR, PistoreseBP, Chi EY, Goodman RB, Matthay MA. Effects of leukotriene B4 in the human lung. Recruitment of neutrophils into the alveolar air spaces without a change in protein permeability. J Clin Invest 1989;84:1609-19. Michel O, Ginanni R, Bon BL, Content J, Duchateau J, Sergysels R. Inflammatory response to acute inhalation of endotoxin in asthmatic patients. Am Rev Respir Dis 1992; 146:352-7. Wang Z, Malmerg P, Larsson P, Larsson BM, Larrson K. Time course of interleukin-6 and tumor necrosis factor-a increase in serum following inhalation of swine dust. Am J Respir Crit Care Med 1996;153:147-52. MulliganMS, VaporciyanA_A,MiyasakaM, Tamatani T, Ward PA. Tumor necrosis factor-a regulates in vivo intrapulmonary expression of ICAM-1. Am J Pathol 1993;142:1739-49.
Inhalation of lipopolysaccharide (LPS) by humans rapidly recruits neutrophils to alveolar structures. Recruitment of neutrophils may be mediated in part by intrapulmonary release of cytokines such as tumor necrosis factor-s, interleukin (IL)- 113,and IL-8, although the kinetics of cytokine accumulation and neutrophil recruitment to the lungs after LPS inhalation have not been determined. Release of some cytokines in response to LPS is reported to be decreased in smokers' alveolar macrophages compared with nonsmokers', suggesting responses to LPS may differ in smokers (S) and nonsmokers (NS). To assess the kinetics of early cytokine accumulation after LPS inhalation and to compare inflammation induced in LPS-exposed S and NS, we performed bronchoalveolar lavage (BAL) in 28 subjects (14 NS and 14 S) at 90 or 240 minutes after inhalation of aerosolized LPS (30 ~g). BAL performed at 90 and 240 minutes after LPS inhalation recovered increased numbers of neutrophils and lymphocytes in both NS and S compared with an unexposed control group (10 NS, 10 S), with greater recovery of neutrophils in S than NS (p < 0.00 I). BAL fluid supemate concentrations of IL-8, IL-113, and tumor necrosis factor-oL at 90 minutes were increased in S and NS compared with an unexposed control group. IL-8 and tumor necrosis factor-~ concentrations were similar in S and NS; however, IL- 113 concentrations were greater in S (p < 0.005). BAL fluid concentrations of IL-113 and IL-8 at 90 minutes correlated with absolute neutrophil recovery in S and NS. These findings suggest that the rapid accumulation of cytokines, particularly IL-113 and IL-8, contributes to lung neutrophil recruitment after LPS inhalation. In addition, parameters of pulmonary inflammation present in S after LPS inhalation are similar to or increased compared with those present in NS. (J Lab Clin Med 1997;129:106-14)
Abbreviations: BAL = bronchoalveolar lavage; ELISA = enzyme-linked immunosorbent assay; IL-8 = interleukin-8; IL-113= interleukin-1 beta; LPS = lipopolysaccharide; TNF-~ = tumor necrosis factor alpha
From the Divisionof Pulmonaryand Critical Care Medicine, Department of Medicine, University of Kansas Medical Center, and Department of Medicine, Department of Veterans AffairsMedical Center. Supported by American Heart Association, Kansas Affiliate, and by the Research Service, Department of Veterans Affairs. Submitted for publication July 15, 1996; revision submitted July 15, 1996; accepted Aug. 1, 1996. Reprint requests: Lewis J. Wesselius, MD, Department of Medicine (111), Department of Veterans Affairs Medical CenterKansas City, 4801 LinwoodBlvd., Kansas City, MO 64128. Copyright © 1997 by Mosby-Year Book, Inc. 0022-2143/97 $5.00 + 0 5/1/77480 106
here is considerable evidence that LPS present in organic dusts is an important mediator of pulmonary inflammation associated with exposure to grain, cotton, and swine dusts. 1'2 Inhalation of LPS by human subjects induces an intense inflammatory response characterized by rapid neutrophil and lymphocyte recruitment to lung structures. 3'4 Inhalation of LPS in sufficiently high doses can induce airway dysfunction, and longer term exposure to smaller amounts of inhaled LPS may cause chronic cough and increased sputum production and may lead to accelerated loss of lung function. 5-8
T
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Previous studies indicate that intrapulmonary instillation of LPS in rats induces the expression of inflammatory cytokines by alveolar macrophages. 9 Inhalation of LPS-containing grain dusts by h u m a n subjects also has b e e n shown to induce expression of IL-8, TNF-c~, and IL-113 by alveolar macrophages. 1° The intrapulmonary accumulation of macrophagederived cytokines such as IL-8 and IL-113 may contribute to early neutrophil recruitment after LPS inhalation in humans, 11-14 although cytokines may also be derived from other cells such as neutrophils, epithelial cells, or fibroblasts, ls-17 Neutrophil recruitment to the lungs occurs within 3 hours of LPS inhalation by h u m a n volunteers, suggesting that cytokine accumulation must occur rapidly to mediate neutrophil recruitment. The early time course of cytokine and neutrophil accumulation within h u m a n lungs after LPS inhalation has not previousb, been determined. Alveolar macrophages recovered from cigarette smokers are reported to release decreased amounts of inflammatory cytokines including I L - I ~ and TNF-oL after in vitro exposure to LPS compared with alveolar macrophages recovered from nonsmokers. ls-2° This observation suggests that the pulmonary inflammatory response to LPS inhalation in smokers may be diminished compared with that in nonsmokers. Clinical observations, however, suggest t h a t cigarette smoking is associated with enhanced risk of pulmonary dysfunction in response to chronic exposures to inhaled L P S Y '22 In this study we compared pulmonary inflammation associated with LPS inhalation in a group of healthy smokers with a group of never-smokers to determine whether there w a s evidence of diminished lung inflammatory responses to inhaled LPS in smokers. METHODS Study subjects and spirometry. The LPS-treated study population consisted of 28 subjects including 14 nonsmokers and 14 current smokers studied at either 90 minutes (7 nonsmokers, 7 smokers) or 240 minutes (7 nonsmokers, 7 smokers) after inhalation of LPS. Smokers had a minimum smoking history of 10 pack years. In addition, we studied 20 matched subjects (10 nonsmokers and 10 smokers) who were not treated with LPS and who served as control groups. No subject in the study was taking medications or reported taking aspirin or nonsteroidal medications within the previous 2 weeks. All subjects denied chronic production of sputum, and none reported a recent history of upper respiratory tract infection. All subjects had baseline spirometry performed with a pneumotachograph (Medical Graphics System 1070, St Paul, Minn.), and the best of three efforts was recorded. All subjects studied 4 hours after LPS inhalation had spirometry re-
Wesselius et al.
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peated immediately before bronchoscopy. The protocol used was approved by the Human Subjects Committee of the Kansas City Department of Veterans Affairs Medical Center. Written informed consent was obtained from all subjects. LPS inhalation. A solution of LPS was made by adding LPS (Escherichia coli, serotype 0111:B4, Sigma, St. Louis, Me.) to sterile normal saline solution. A solution containing 30 mg LPS in 3 ml saline solution was inhaled by subjects over approximately 15 minutes with a nebulizer (Respirgard II, Marquest, Englewood, Colo.) and a Pulmo-Aid machine. This dose of inhaled LPS was chosen on the basis of preliminary studies demonstrating neutrophil recruitment in the absence of a significant reduction in FEV 1 and is consistent with previous studies. 5 Bronchoscopy and bronchoalveolar lavage. Subjects received light sedation with intravenous meperidine. After the oropharynx was anesthetized with tetracaine, brenchoscopy was performed by the oral route. Bronchoalveolar lavage was performed in subsegments of the middle lobe and lingula; 6 aliquots of 20 ml of sterile 0.9% saline solution were instilled for a total of 120 ml lavage in each lung. Lung lavage fluid was recovered by gentle suction and was sequentially processed with the initial 20 ml aliquot (bronchial sample) collected separately from the following 4 aliquots (alveolar aliquots), which were pooled for analysis. Processing of specimens. BAL fluid was immediately filtered over sterile gauze to remove mucous. Cells were recovered by centrifugation (400g), and aliquots of BAL fluid were decanted and stored at - 7 0 ° C until subsequent analysis was performed. Cell recovery was determined with a hemacytometer, and a cell differential was determined by counting 200 cells on a Giemsa/Wright-stained cytocentrifuge preparation. Cytokine and protein determinations. The concentrations of IL-8, TNF-c~, and IL-113 present in bronchoalveolar lavage supernates and serum obtained at the time of bronchoscopy were determined with a standard ELISA (R&D Systems, Minneapolis, Minn.) with antibodies raised against human recombinant IL-8, TNF-a, and ILl[3. The sensitivity of the ELISA assays used is as follows: IL-8 assay is sensitive to 3 pg/ml, the TNF-c~ assay is sensitive to 4.4 pg/ml, and the IL-l[3 assay is sensitive to 0.3 pg/ml. We ran all samples in duplicate. The ELISA assays we used are not reported to exhibit detectable cross-reactivity with the other cytokines we tested. Protein content of BAL fluid was determined with a bicinchoninic acid protein assay reagent (BCA kit; Pierce, Rockford, Ill.) with bovine serum albumin used as a standard. Statistical analysis. Data are expressed as mean +_ SE. Differences between groups were analyzed with the Mann-Whitney rank sum test. In all tests statistical significance was identified at the p < 0.05 level. We conducted least squares linear regression analysis to examine the relationships between BAL fluid supernate content of IL-8 and IL-113 after LPS inhalation and numbers of neutrophits in recovered cells.
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Table I. C h a r a c t e r i z a t i o n
of study populations
Smoking history (pack yeors)
FEV1% pred/ FVC % pred
36.3 _+ 2.2
0
38.1 _+ 1.6
27 _+ 3
102 _+2 104 _+ 2 92_+4 104 -+ 2
LPS-Exposed 90 minutes Nonsmokers (n = 7)
36.4 _+ 2.1
0
Smokers (n = 7)
40.8 _+ 2.3
34 -+ 4
34.0 _+ 2.4
0
38.7 + 2.7
31 _+ 4
Age (yr) Control group Nonsmokers (n = 10) Smokers (n = 10)
240 minutes Nonsmokers (n = 7) Smokers (n = 7)
98_+3 99_+2 88 _+4 101 -+2 101 103 91 102
-+3 _+ 3 _+4 __ 3
Values expressed as mean _+SE.
Table II. C e l l a n d p r o t e i n r e c o v e r y in BAL f l u i d Total cells (× 106) No LPS Nonsmokers Smokers After LPS 90 minutes Nonsmokers Smokers 240 minutes Nonsmokers Smokers
Alveolar macrophage (× 10n/ml)
Neutrophils (x 104/mi)
Lymphocytes (x 104/mi)
Protein (mg/ml)
11.7 + 0.8 26.4* -+ 3.0
7.9 -+ 0.5 19.1" _+ 3.1
0.03 _+ 0.01 0.24 + 0.06
0.38 -+ 0.10 0.44 -+ 0.15
.088 -+ .010 .119 -+ .019
13.3 -+ 1.1 30.3* -+ 3.7
8.5 -+ 0,4 25,5" + 3.7
1.621- -+ 0,6 4.51" _+ 1.28
0.47 + 0.15 0.61 _+ 0.13
.117 -+ .018 .138 -+ .019
17.3 + 2.4 47.2* + 13.9
11,11- + 1.3 31.8" -+ 7.0
2.131- -+ 0.52 7.05* _+ 2.86
0.811- _+ 0.18 0.961- + 0.20
.127 -+ .010 .143 + .021
*p < 0.05 compared with nonsmokers. 1-P < 0,05 compared with no LPS.
RESULTS Subject characteristics and spirometry. The charac-
teristics of the study populations are provided in Table I. All of the smoking subjects were current smokers and had at least a 10 pack year total smoking history (range 10 to 50 pack years). Smoking histories and ages were similar in the control group (not LPS-exposed) and in subjects studied 90 or 240 minutes after LPS inhalation. Mean baseline spirometric measurements for each group are also provided in Table I. The mean FEV1 was lower in smoking subjects than in nonsmokers; however, all smokers had an FEV1 that was greater than 70% of the predicted value. Spirometry performed just before bronchoscopy in all subjects studied 4 hours after LPS inhalation demonstrated that
none of the subjects had a significant (>10%) decrease in FEV1 compared with baseline (data not shown). BAL fluid cellularity. Bronchoalveolar lavage performed in subjects without exposure to LPS recovered significantly more cells in smokers than in nonsmokers primarily as a result of increased recovery of alveolar macrophages (Table II). Subjects studied after LPS inhalation had an increase in total cell recovery at both 90 and 240 minutes, which was a result of increased recovery of both neutrophils and alveolar macrophages. Significantly greater numbers of neutrophils were recovered by BAL in smokers at both 90 and 240 minutes after LPS than in nonsmokers (Table II). Also, significantly greater numbers of lymphocytes were recovered by bronchoalveolar la-
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2800-
2400-
2000-
IL-8 pg/ml 1600-
1200-
OO
800-
400-
_~.
o o0
%.
NS S p<.05 NO LPS
NS
S
NS I
n.s. 90 min
I S I
n.s.
240 min TIME AFTER LPS
Fig. 1. Concentrations of IL-8 in B A L tluid supernates recovered from groups either exposed to no LPS (control) or groups studied at 90 or 240 minutes after inhalation of LPS (30 rag). * = p < 0.05 compared with concentrations at 90 minutes, n.s. = not significantly different.
vage in both smokers and nonsmokers at 240 minutes after inhalation of LPS with similar increases in both groups (Table II). BAL fluid concentrations of cytokines and protein.
Evaluation of BAL fluid supernatants recovered from subjects without exposure to LPS demonstrated a small but significant increase in IL-8 concentrations in smokers compared with nonsmokers (p < 0.05) (Fig. 1). In several smokers BAL fluid supernatant concentrations of IL-I[3 were also greater than values present in nonsmokers; however, differences between groups were not significant (Fig. 2). BAL fluid supernatant concentrations of TNF-~ were similar in both smokers and nonsmokers (Fig. 3). The total protein content of BAL fluid was slightly higher in smokers than in nonsmokers as has been noted in previous studies.23 Concentrations of IL-8, TNF-oL, and IL-113 in recovered BAL fluid were increased at 90 and 240 minutes after LPS inhalation in both nonsmokers
and smokers compared with respective unexposed groups (Figs. 1, 2, and 3). Concentrations of IL-8 and TNF-a were not significantly different in smokers studied 90 or 240 minutes after LPS inhalation compared with nonsmokers. In contrast, BAL fluid concentrations of IL-I[3 were significantly increased in smokers compared with nonsmokers in subjects studied both at 90 and 240 minutes after LPS (Fig. 3). Concentrations of TNF-oL and IL-113 in BAL fluid supernates were not significantly different at 240 minutes compared with 90 minutes after LPS inhalation in either smokers or nonsmokers. In contrast, concentrations of IL-8 were significantly lower at 240 minutes compared with 90 minutes (0 < 0.05) in both smokers and nonsmokers. Protein concentrations in BAL fluid after LPS inhalation were increased in both smokers and nonsmokers compared with the unexposed control group at 90 minutes; however, differences were not significant (Table II). A small but significant in-
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120 O0
100
0
O0
e
TNF pg/ml
80
eeee
0
o
O0 0
60'
O0
0
ee
40-
0
o
ee
20-
•
oooo
ee
oo
m=aa
I
nnn~
NS '
S
I
I
n.s.
NO LPS
NS
I
I
S I
NS I
I
n.s,
n.s.
90 min
I S
240 min TIME AFTER LPS
]Fig. 2. Concentrations of TNF-a in BAL fluid supernates recovered from groups either exposed to no LPS (control) or groups studied at 90 or 240 minutes after inhalation of LPS (30 mg). n.s. = not significantly different.
crease occurred in total protein content of BAL fluid at 240 minutes after LPS inhalation in both smokers and nonsmokers (Table II). No difference was seen in the absolute change in BAL fluid protein content in smokers or nonsmokers. Serum cytokine concentrations. S e r u m c o n c e n t r a -
of TNF-oL and IL-113 were determined in unexposed subjects and 90 or 240 minutes after LPS inhalation in exposed subjects. In unexposed subjects serum concentrations of TNF-oLwere undetectable and serum concentrations of IL-I[3 were low or undetectable. At 90 and 240 minutes after LPS inhalation serum TNF-oL was detectable in 23 of 28 subjects, and values were similarly increased in smokers and nonsmokers (Table III). Serum concentrations of IL-113were similarly increased in both groups at 90 and 240 minutes after LPS inhalation compared with unexposed subjects (Table III). Correlations. To assess whether there was a relationship between IL-113 concentrations in BAL fluid and lung neutrophil recruitment, we evaluated for correlations between these values in smokers and nonsmokers. Statistical analysis demonstrated a cortions
relation between IL-l[3 concentrations in BAL fluid supernates recovered at 90 minutes after LPS inhalation and the numbers of neutrophils in cells recovered by BAL (Fig. 4). The correlation between IL-113 concentrations and absolute neutrophil recovery (r = .71, p < 0.05) was slightly greater than the correlation between IL-8 concentrations and absolute neutrophil recovery (r = 0.64, p < 0.05). DISCUSSION
The data obtained in this study indicate that there are marked increases in alveolar concentrations of IL-8, IL-I[3, and TNF-R in human subjects within 90 minutes of inhalation of LPS, supporting the concept that these cytokines contribute to early lung neutrophil recruitment induced by LPS. In addition, in this study we studied both smokers and nonsmokers after LPS inhalation and found that both groups had similar BAL fluid concentrations of IL-8 and TNF-~, but smokers had increased concentrations of IL-I[3 compared with nonsmokers. Recovery of neutrophils by BAL after LPS inhalation was also greater in smokers than nonsmokers, indicating that
J L a b Ctin M e d V o l u m e 129, N u m b e r
1
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111
160-
140-
120-
100-
1L- ll] pg/ml
80-
60-
O
40-
o
20o
•. "" NS
e
_~_
!
S
NS
I
I .... n.s.
I
..
I
S
I
NO LPS
NS
I
I
I
S 1
p<.005
p<.005
90 min
240 min TIME AFTER LPS
Fig. 3. Concentrations of IL-113 in BAL fluid supernates recovered from groups either exposed to no LPS (control) or groups studied at 90 or 240 minutes after inhalation of LPS (30 mg). n.s. = not significantly different.
Table III. TNF-= a n d IL-113 c o n c e n t r a t i o n s in serum ( p g / m l ) No LPS* TNF-c~
Nonsmokers Smokers
ND ND
90 Minutes I L- I I~
0.3 -+ 0.1 0.3 -+ 0.1
240 Minutest
TNF-c~
I L- 113
TNF-~
92:1: -+ 19 805 -+ 15
1.3:1: -+ 0.3 1.45 -+ 0.2
81:1: + 15 655 -+ 6
IL- 113
5.0:1: +- 0.2 3.1:1: -+ 0.4
*p < 0.05 compared with No LPS group. 1-n = 7 for 90 and 240 minute groups in nonsmokers and smol, ers. *n = 10 for No LPS group in nonsmokers and smokers. ND, Not detected in any subject.
pulmonary inflammation associated with LPS inhalation is not diminished in smokers compared with nonsmokers. In our studies of control (not LPS-exposed) smokers and nonsmokers we found evidence of pulmonary inflammation associated with smoking alone including increases in BAL fluid concentrations of IL-8 and increased recovery of neutrophils in smokers compared with nonsmokers, findings that are
consistent with previous studies, ls'24 Because we did not determine baseline neutrophil recovery or cytokine concentrations in BAL fluid for each LPSexposed subject, we cannot determine a percentage increase in these parameters for individual subjects and therefore cannot determine with certainty whether the effects of LPS inhalation are additive or synergistic with smoking exposures in the induction of inflammation. Concentrations of IL-I~ in BAL
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!60 140 120 100 L_
°
•
It-113
pg/ml
80 604020
J
O 9/0
p < .05
O • o
I
I
I
I
I
I
t
I
I
I
I
I
1
2
3
4
5
6
7
8
9
10
11
12
Neutrophil Recovery x104/ml
Fig. 4. Relationshipbetween concentrationof IL-113in BAL fluidsupernates and absolute numbers of neutrophilsin cellsrecoveredby BAL.Values are fromgroups studiedat 90 minutesafterLPS inhalation and includenonsmokers(open circles) and smokers (closed circles).
fluid of LPS-exposed nonsmokers were sixfold greater than in unexposed nonsmokers, whereas IL-l[3 concentrations were approximately twelvefold greater in LPS-exposed smokers than in unexposed smokers. This finding suggests the possibility that intrapulmonary accumulation of IL-113 after LPS inhalation may be enhanced in some smokers. Because our smoking population was slightly older than the nonsmoking population and had lower FEV 1 values, these variables could also have contributed to the differences we observed. The accumulation of proinflammatory cytokines in the lower respiratory tract after LPS exposure has been proposed to contribute to lung inflammation and injury associated with LPS exposures) ° Previous studies indicate that intrapulmonary administration of IL-8 or IL-I[3 in experimental animals induces substantial recruitment of neutrophils to the lungs, so the accumulation of both of these cytokines within the lungs by 90 minutes after LPS inhalation supports the concept that these cytokines contribute to early LPS-induced recruitment of neutrophils to the lungs./1'13 Because IL-8 is also chemotactic for
lymphocytes, increased lung content of this cytokine may contribute to the accumulation of lymphocytes after LPS inhalation, which we noted, and which is similar to findings of previous studies.3'4'25 The presence of increased lung concentrations of IL-l[3 within alveolar structures after LPS inhalation may contribute to greater neutrophil recruitment by inducing enhanced expression of adhesion molecules on lung endothelial and epithelial cells, which could promote neutrophil recruitment. 26'27 The relatively greater concentrations of IL-113 present within the lungs of smokers may contribute to greater absolute numbers of neutrophils recovered from LPS-exposed smokers compared with nonsmokers. It is possible that differences in other neutrophil chemoattractants such as leukotriene 134 or GRO alpha may also contribute to differences in neutrophil recruitment. 2s'29 In addition to elaborating neutrophil chemoattractants, alveolar macrophages also release a low-molecular weight factor that inhibits neutrophil chemoattractant responses. 3° Diminished release of this inhibitory factor by smokers' alveolar macrophages could also poten-
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tially contribute to increased lung neutrophil recruitment in smokers. The finding of increased lung concentrations of IL-113 in smokers compared with nonsmokers after LPS inhalation is somewhat surprising, because alveolar macrophages recovered from smokers release substantially less IL-I[3 in response to in vitro LPS exposure than alveolar macrophages from nonsmokersJ 9'2° It is possible, however, that the greater number of alveolar macrophages present in smokers leads to a greater respiratory tract burden of alveolar macrophage-derived IL-113 after LPS exposures. Alterr~atively, IL-I~ may be derived from other cells such as neutrophils, which can be a source of IL-I[3 after endotoxin exposures. 31 Previous studies have demonstrated that human alveolar macrophages stimulated with LPS in vitro do not release significant amounts of IL-I[~ during the initial 4 hours after stimulation. 14 Our finding of IL-I[~ accumulation by 90 minutes after LPS inhalation suggests that either alveolar macrophage elaboration of IL-I[3 occurs more rapidly in vivo or that IL-113 accumulating initially after LPS inhalation is derived from cells other than alveolar macrophages. Although LPS inhalation was associated with recruitment of neutrophils to lung structures and increased lung concentrations of inflammatory cytokines in both smokers and nonsmokers, there was only a limited effect on lung vascular permeability. This finding is similar to a previous report in which leukotriene B4-induced lung neutrophil recruitment in human lungs was not associated with increased lung vascular permeability. 32 The absence of significant increases in lung vascular permeability despite increases in lung content of TNF-c~ and IL-l[3 suggests that the lung epithelial surface in humans is relatively resistant to injury from these cytokines at least at the concentrations induced in these studies. Serum concentrations of TNF-a and IL-l[3 were increased by 90 minutes after LPS inhalation, indicating a systemic inflammatory response was induced by LPS inhalation. These findings extend previous observations that LPS inhalation increased serum concentrations of TNF-c~ at 60 minutes after exposure in asthmatics and are consistent with recent reports on the time course of increased serum TNF-a after inhalation of LPS-containing organic dusts. 33'34 Increases in serum TNF-o~ may contribute to lung recruitment of neutrophils after LPS inhalation by inducing expression of adhesion molecules on lung endothelial cells. 35 In summary, this study indicates that inhalation of LPS by human subjects induces rapid accumulation of IL-8, IL-I~, and TNF-cx within the lower respira-
Wesselius et al.
113
tory tract. The kinetics of accumulation of IL-113 and IL-8 within alveolar structures after LPS inhalation are consistent with a role for both cytokines in early LPS-induced lung neutrophil recruitment. It is interesting that the accumulation of IL-lJ3 occurs more rapidly than is required for in vitro elaboration of IL-I[~ by LPS-stimulated alveolar macrophages. Comparison of pulmonary inflammation induced by LPS inhalation in smokers and nonsmokers indicates that BAL in LPS-exposed smokers recovered greater numbers of neutrophils and that recovered BAL fluid had greater concentrations of IL-I[3 compared with nonsmokers. The higher BAL fluid concentrations of IL-113 present in BAL fluid recovered may be a result of either additive inflammatory" effects of smoking and LPS exposure or an interaction between smoking and LPS exposure with regard to accumulation of intrapulmonary IL-I[3. The authors thank Dr. James Williams, Jr. for helpful suggestions in this study, and LaTrisha Gaston for her assistance in the preparation of the manuscript.
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