effects cigarette smoke components on in vitro chemotaxis ...phenolphthalein glucuronide was...

INFECTION AND IMMUNITY, Apr. 1977, p. 240-248Copyright ©D 1977 American Society for Microbiology

Vol. 16, No. 1Printed in U.S.A.

Effects of Cigarette Smoke Components on In VitroChemotaxis of Human Polymorphonuclear LeukocytesRAYMOND B. BRIDGES,* J. H. KRAAL, L. J. T. HUANG, AND M. B. CHANCELLOR

Department of Oral Biology and Periodontology, College of Dentistry, University of Kentucky, Lexington,Kentucky 40506

Received for publication 4 November 1976

Some ciliostatic components ofcigarette smoke were studied as inhibitors of invitro chemotaxis of human polymorphonuclear leukocytes (PMNs). In compari-son to their concentration in an inhibitory level of cigarette smoke, the unsatu-rated aldehydes acrolein and crotonaldehyde were the most potent inhibitors,whereas nicotine, cyanide, acetaldehyde, and furfural were the next strongestinhibitors. In contrast, sulfide, propionaldehyde, butyraldehyde, and the phe-nols (phenol and o-, m-, and p-cresol) were relatively weak inhibitors of PMNchemotaxis. Acrolein and crotonaldehyde mimicked whole cigarette smoke intheir effects on PMNs by not causing loss ofPMN viability, yet their effects wereprevented by the addition of cysteine. On the other hand, addition of nicotine,cyanide, acetaldehyde, and furfural to PMN suspensions resulted in a limitedloss of cellular viabilities, and their effects on PMNs were not prevented bycysteine. Of the tested components, only cyanide significantly altered PMNglucose metabolism by increasing carbon flow via the glycolytic and hexosemonophosphate pathways in a manner similar to that observed with wholecigarette smoke. The results of this study suggest that the unsaturated alde-hydes, including acrolein and crotonaldehyde, are major contributors to theinhibitory properties of cigarette smoke. The inhibitory effects of these unsatu-rated aldehydes are probably due to a direct interaction of these oxidants and/orthiol-alkylating agents with PMNs, yet the glucose metabolism of these cells isunaffected. One interpretation of these data is that PMN chemotaxis is depend-ent upon particular cellular proteins containing one or more essential thiolgroup(s) but that these proteins are unrelated to glucose metabolism.

The function of human polymorphonuclearleukocytes (PMNs) is severely affected by ciga-rette smoke both in vivo and in vitro. The mo-tility and oxygen consumption of human oralPMNs from smokers were inhibited as com-pared to oral PMNs from nonsmokers (14).Also, the chemotaxis of peripheral blood leuko-cytes has been shown to be depressed in sub-jects who smoke as compared either to the samesubjets abstaining from smoking or to non-smokers (23).

In a more recent study (10), the water-solublefraction of cigarette smoke, whole tobaccosmoke, and the gas phase of smoke were shownto be potent inhibitors of in vitro PMN chemo-taxis. Whole smoke was more inhibitory thangas phase, suggesting the possibility that theinhibitory components of smoke were associ-ated with both the particulate and gas phasefractions. However, none of the tested tobaccosmoke products at concentrations inhibitory tochemotaxis were found to have a cytotoxic ef-fect on PMNs, as measured by trypan blue

exclusion or enzyme (i.e., lactic acid dehydro-genase [LDH] or ,8-glucuronidase) release. Fur-thermore, these tobacco smoke products did notinhibit glucose metabolism in PMNs, butrather increased (twofold) glucose catabolismvia both the glycolytic and hexose monophos-phate pathways. Finally, cysteine was shownto afford complete protection against the inhibi-tion of PMN chemotaxis by the water-solublefraction but only partial protection against theinhibitory effects of whole smoke and the gasphase of smoke.

In the present study, we have tested compo-nents of cigarette smoke previously shown tocause cilia toxicity (26) for their ability to in-hibit PMN chemotaxis. The aim of this studywas to determine which of these componentswas most deleterious to PMN function.

MATERIALS AND METHODSIsolation of PMNs. PMNs were isolated from pe-

ripheral blood of nonsmoking, healthy, male volun-teers who were fasted at least 12 h before venipunc-

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EFFECTS OF CIGARETTE SMOKE COMPONENTS 241

ture. PMNs were separated from lym-iphocytes anderythrocytes by dextran sedimentation of heparin-ized whole blood (11), Hypaque-Ficoll gradient cen-trifugation (9), and hypotonic saline lysis of residualerythrocytes (11). PMNs were resuspended in Geymedium containing 2% bovine serum albumin andbicarbonate to give 3 x 106 PMNs per ml. Thisprocedure produced PMN populations of greaterthan 98% purity with greater than 98% viability asmeasured by trypan blue exclusion (25).

Pure substances of cigarette smoke. The com-pound to be tested was dissolved in Gey medium (100AD) or dimethyl sulfoxide (10 ,lI) and added to PMNsuspensions (1 ml), each in amounts to give the finalconcentrations indicated. An equivalent amount ofsolvent was added to control PMN suspensions.These solutions were freshly prepared on the day ofthe experiment, and the pH of the compounds pre-pared in Gey medium was adjusted to 7.4, if neces-sary. Dimethyl sulfoxide was an acceptable solventfor some compounds, since it did not affect in vitroPMN chemotaxis, confirming the previous observa-tion of Borel (6).

Cigarette smoke components were added to PMNsuspensions in final concentrations that were previ-ously shown to result in cilia toxicity (26). If thisconcentration of agent was found either not to affector to inhibit PMN chemotaxis, the concentration ofagent was then increased or decreased in 10-foldincrements, respectively. In this manner, the rangeof agent concentration necessary to produce PMNchemotaxis inhibition was approximated. Fromthese data, components for further study were se-lected on the basis of their relative concentration intobacco smoke as compared to the concentration in-hibitory to chemotaxis and their limited cytotoxiceffects on PMNs. With the demonstration of mini-mal cytotoxic effects, the effects of each componenton PMN glucose metabolism were then studied. Fi-nally, cysteine was tested as a protective agentagainst the inhibitory action of each of these selectcomponents, since cysteine should provide at leastsome protection against the inhibitory effects ofthese components, ifindeed they mimic the effects ofwhole tobacco smoke (10).

Chemotaxis assay. Chemotaxis experiments weredone using a modified Boyden chamber (8). To avoiderrors introduced by diminished adherence ofPMNs, two membrane filters (mesh sizes, 0.45 and 3,tm; Millipore Corp., Bedford, Mass.) were used toseparate the two compartments of the modified Boy-den chamber (18). Fresh, human, autologous serumactivated with endotoxin was prepared as describedpreviously (10) and layered in the lower compart-ment of the Boyden chamber adjacent to the smallermesh size (0.45 ,m) Millipore filter. The upper com-partment was filled with cell suspension (3 x 106PMNs per ml) with or without the addition of testagent. The chambers were incubated at 37°C for 2 hin an atmosphere of humidified air containing 5%carbon dioxide. After incubation, the Millipore fil-ters were removed, stained with Ehrlich hematoxy-lin, cleared, and mounted on microscopic slides (8).Cells were counted on the attractant side of the 3-,m filter in 10 consecutive evenly spaced fields as

defined by an ocular grid. Chemotaxis assays weredone in duplicate or triplicate. The chemotaxis re-sults are expressed as a ratio of experimental tocontrol. A numerical value for this ratio of 1 represented stimulation of chemotaxis.

In experiments testing the protection of cysteineagainst inhibition of PMN chemotaxis, cysteine (fi-nal concentration, 10 mM) was added prior to theaddition of the cigarette smoke constituent.

Effects of cigarette smoke components on PMNintegrity. The test agents, at concentrations inhibi-tory to in vitro PMN chemotaxis, were incubatedwith PMN suspension (3 x 10" cells per ml) in Geymedium for 2 h at 37°C. The viability of the treatedPMN was measured by the exclusion of trypan bluedye, as previously described (25). The number ofPMNs per milliliter of suspension was also quanti-tated in the treated cellular suspensions to insurethat there were no cellular losses. The PMNs wereremoved from the remaining suspension by centrifu-gation, and the cell-free supernatants, collected bydecantation, were analyzed for LDH (EC 1.1.1.27)and /3-glucuronidase (EC 3.2.1.31) activities.As suitable controls, PMNs in an equivalent sus-

pension (3 x 106 PMNs per ml) in Gey medium weredisrupted by treatment with a Branson Sonifier(W140) at maximal voltage output for two 30-sbursts. After centrifugation, the supernatants fromthe disrupted cell suspension were collected by de-cantation and incubated in the presence and absenceof test agents for 2 h at 37°C. The treated and un-treated supernatants were analyzed for LDH and 1-glucuronidase activities. LDH activity of the super-natant in the absence of test agent was taken as anindex of complete cellular lysis. Since the f8-glucu-ronidase activity of these supernatants representeda constant proportion (65%) of the total 83-glucuroni-dase activity of these cells, as obtained by sonictreatment in the presence of Triton X-100 (PackardInstrument Co., Inc., Downers Grove, Ill.), this en-zyme activity was used as an index of lysosomalenzyme release. Furthermore, these data indicatedthe effects of the test agents on these enzymaticactivities.The assay for LDH activity was done as previ-

ously described (5). The oxidation of reduced nico-tinamide adenine dinucleotide was followed contin-uously at 340 nm using a Unicam SP 1700 ultravioletspectrophotometer equipped with a Unicam AR 25linear recorder. A unit ofLDH activity is defined asthe amount of enzyme required to catalyze the oxi-dation of 1 gmol of nicotinamide adenine dinucleo-tide per min.

,B-Glucuronidase activity was measured as previ-ously described (15). Phenolphthalein glucuronidewas incubated with cell supernatants or cell-freesonic extracts for 18 h at 37°C. A unit of /8-glucuroni-dase activity is defined as the amount of enzymerequired to liberate 1 ,umol of phenolphthalein.

Glucose metabolic studies. Glucose metabolismof PMNs in the presence and absence of cigarettesmoke constituents was studied by the previouslydescribed methods (10). Cigarette smoke constitu-ents in 0.3 ml of Gey medium were added to cellular

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242 BRIDGES ET AL.

suspensions (3 ml, 3 x 106 PMNs per ml in Geymedium containing 1 g of glucose per liter). To startthe reactions, 5 ,ul of [1_14C]_ or [6-'4C]glucose (0.5,uCi, 50 ,Ci/,umol) was added. After incubation for90 min at 37°C, the reactions were stopped by acidifi-cation, and the "4CO2 released was trapped andquantitated. The 14CO2 released from incubationmixtures containing either [1-_4C]- or [6-"4C]glucosewas taken as an index of glucose metabolism viaeither the hexose monophosphate pathway or theglycolysis/oxidative metabolism, respectively.PMNs were collected from the acidified reaction

mixture by centrifugation and analyzed for glycogencontent by the method of Anderson and Stowring(1). The deproteinized incubation mixtures werethen analyzed for glucose and lactic acid by usingglucose oxidase (Worthington Biochemicals Corp.,Freehold, N.J.) and the method of Barker and Sum-merson (3), respectively.

Glucose consumed by PMNs was calculated by thedifference between the nanomoles present in incu-bation mixtures without PMNs and the nanomolespresent in test incubation mixtures. Some testagents (acetaldehyde, propionaldehyde, butyralde-hyde, acrolein, and crotonaldehyde) produced an in-terfering colorimetric reaction in the lactic acid de-termination. In these instances, blanks containingan equivalent concentration of test agent were used.

Glucose utilization or lactic acid production wereexpressed as nanomoles of glucose utilized or lacticacid produced per 90 min per 9 x 10V PMNs, respec-tively. The expression of metabolic results in termsof PMN number was previously shown to be valid(10), since equivalent PMN suspensions had a con-stant protein concentration.

Although the metabolic results were highly re-producible within a given experiment, some varia-bility in results was observed with different leuko-cyte preparations. Therefore, metabolic results wereexpressed as a ratio of the experimental values tothe control values. A numerical value for the ratio of>1 represented metabolic stimulation, whereas anumerical value for the ratio of < 1 represented met-abolic depression.

Statistical analysis of data. Chemotactic and glu-cose metabolic data were subjected to statisticalanalysis by using Student's t test, testing the hy-pothesis that the ratio of treated to control wasequal to 1.

RESULTS

Chemotaxis and glucose metabolism of un-treated PMNs. The mean values for chemo-taxis and glucose metabolism of untreatedPMNs are shown in Table 1. Since the glycogencontent of PMNs was negligible (data notshown), the Gey medium was the sole source ofglucose. The extent of PMN glucose metabo-lism via glycolysis (712 nmol) was approxi-mately 130-fold greater than via the hexosemonophosphate pathway (5.6 nmol). The glu-cose catabolic rate via oxidative metabolism asmeasured by 14CO0, released from [6-'4C]glucose

was negligible and unaffected by the testagents reported in this study (data not shown).Effect of cigarette smoke constituents on

PMN chemotaxis. Figure 1 shows the effects ofvarious concentrations of cyanide, sulfide, nico-tine, crotonaldehyde (2-butenal), and acrolein(2-propenal) on PMN chemotaxis. The additionof increasing concentrations of these compo-nents caused a progressive suppression ofPMNchemotaxis. As indicated by the standard er-rors, a high degree of variability in the resultswas generally obtained with concentrations ofagent yielding less than 50% suppression ofchemotaxis.The concentrations of cyanide, sulfide, nico-

tine, crotonaldehyde, and acrolein required toreduce the chemotactic responsiveness to 50% ofcontrol levels was approximately 3.5 mM, 6.5mM, 3.5 mM, 40 uM, and 15 AM, respectively.Thus, among these components, the unsatu-rated aldehydes (i.e., acrolein and crotonalde-hyde) were the most potent inhibitors of PMNchemotaxis. For comparative purposes, the ef-fects of other aldehydes of cigarette smoke aregiven in Table 2. These saturated aldehydes(i.e., acetaldehyde, propionaldehyde, and bu-tyraldehyde) were shown to be much less effec-tive inhibitors of in vitro PMN chemotaxisthan the corresponding unsaturated analogues.Comparison of the concentrations of these com-pounds necessary to inhibit chemotaxis withtheir respective concentrations in an inhibitorylevel of whole tobacco smoke was not favorable,making complete dose-response curves for thesecomponents unnecessary. The concentrations ofthe saturated aldehydes (i.e., propionaldehydeand butyraldehyde) necessary to inhibit PMNchemotaxis were at least 200-fold greater thanthe concentrations of their respective unsatu-rated analogues (i.e., acrolein and crotonalde-hyde). Furfural (2-furaldehyde) was also shown

TABLE 1. Chemotaxis and glucose metabolism ofuntreated PMNs

Measured parameter Mean + SEaChemotaxis (PMNs per 10 fields) 1,528 + 142 (38)b

Glucose utilization (nmol of glu- 871 + 35 (24)cose utilized)"

Lactic acid produced (nmol of lac- 1,423 ± 64 (24)tic acid produced)"

Hexose monophosphate activity 5.6 + 0.4 (24)("'CO2 from [1-"4C]glucose,nmol)'

"SE, Standard error.Number of experiments.

'"Metabolic results are expressed in the units indicatedper 9 x 10' PMNs per 90 min of incubation.

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1j20 4 6 8 10 1 2 46880-

C° 60 - 60 -

0 *

1 2 4 6 8 10 01 2 4 6 8 10

CYNICOIDE(W SUCOO LFDEHYE(m,UM1)

E..T

°0 80 \

C> 1

o \

,,360 0 60 -

M 40 0

~20-20 -

20 _

132 4 6.8 10 120 40 608010NICYINIE (m SUOTALFDEHE(mM)

100c.10

-j~~~~~~~~~0C>0 80

I-

200 2C 02 4 680100 1200 400 608010NICOTIE(mM)CROTONLDEHYE((MM

FIG. 1. Effects of tobacco smoke components on in vitro chemotaxis of PMNs isolated from humanperipheral blood. The results represent the mean percentages of control (+,- standard error) of three to sixseparate e-xperiments. * Mean percentages significantly (P < 0.05) different from control values.

to be a rather weak inhibitor of in vitro PMN trations of these phenolic compounds in excesschemotaxis.- of 5 mM were required to cause a significantPhenol and o-, m-, and p-cresol were also (P < 0.05) inhibition ofPMN chemotaxis (data

weak inhibitors of PMN chemotaxis. Concen- not shown).

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TABLE 2. Effects of the saturated aldehydes of cigarette smoke on PMN chemotaxisTest agent Concn (mM) Ratio to controls (± SE) pa

Acetaldehyde 0.67 0.965 ± 0.167 (6)b NS6.7 0.422 ± 0.029 (3)


TABLE 4. Effects of cigarette smoke constituents on PMN glucose metabolism

Parameter measured Ratio to control t SE

Nicotine (10 mM)

Sulfide (10 mM)

Cyanide (10 mM)

Acetaldehyde (67 mM)

Acrolein (0.2 mM)

Propionaldehyde (60 mM)

Crotonaldehyde (0.1 mM)

Butyraldehyde (90 mM)

Furfural (78 mM)

Glucose utilizedLactic acid producedHMPC activity

Glucose utilizedLactic acid producedHMP activity








0.92 ± 0.18 (6)b1.34 ± 0.17 (6)0.95 ± 0.10 (6)

1.48 ± 0.33 (4)1.09 ± 0.12 (4)1.29 + 0.14 (4)

2.21 0.10 (9)1.84 ± 0.06 (9)3.88 ± 0.66 (9)

0.94 ± 0.05 (4)0.89 ± 0.08 (4)1.21 ± 0.42 (4)

1.35 ± 0.40 (5)0.73 ± 0.13 (5)1.66 ± 0.39 (5)

1.08 ± 0.10 (4)1.46 ± 0.28 (4)1.01 0.15 (4)

0.91 0.15 (4)0.97 ± 0.08 (4)1.21 ± 0.30 (4)

0.80 + 0.13 (4)1.52 0.78 (4)1.56 ± 0.85 (4)

1.01 ± 0.07 (4)1.04 + 0.04 (4)2.51 ± 1.33 (4)

" P, Probability that the ratio of experimental to control is equal to 1. NS, Nonsignificance at the 0.05level.

" Number of experiments.HMP, Hexose monophosphate.

10 mM cysteine. Thus, cysteine provided com-plete protection to PMN against inhibitory con-centrations of acrolein and crotonaldehyde.However, cysteine did not afford any protectionagainst inhibitory concentrations of cyanide,sulfide, nicotine, acetaldehyde, propionalde-hyde, butyraldehyde, and furfural (data notshown).

DISCUSSIONThe previous demonstration that in vitro

PMN chemotaxis was inhibited by cigarettesmoke or its fractions (10) suggested a study ofthe particular.constituents of cigarette smokeand their effects on PMN chemotaxis. From thedata derived from this previous study (10), cri-teria were established for the selection of theconstituent (or class of constituents) contribut-ing most to the inhibitory effects of whole ciga-

rette smoke. The criteria were establishedwithout consideration of additive, synergistic,or antagonistic effects that may occur with mix-tures of cigarette smoke constituents. Thesecriteria are as follows: (i) the concentration ofconstituent found to be inhibitory to chemo-taxis should closely approximate its concentra-tion in an inhibitory level of whole cigarettesmoke; (ii) the constituent at a concentrationinhibitory to PMN chemotaxis should not affectPMN integrity, if the effects of the constituentmimic those of whole cigarette smoke; and (iii)the inhibition ofPMN chemotaxis by a particu-lar component should be at least partially pre-vented by cysteine, since cysteine protectedagainst cigarette smoke inhibition of PMNchemotaxis (10). Moreover, the effects of eachconstituent on PMN glucose metabolism werestudied to deternine if there was any correla-

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Treatment pa

NSNSNS

NSNSNS

246 BRIDGES ET AL.

tion between the twofold stimulation of PMNglucose metabolism observed with cigarettesmoke exposure (10) and the agent(s) responsi-ble for chemotaxis inhibition.The inhibition of PMN chemotaxis by the

components tested in this study was probably aresult of the direct interaction with the cells,since each of the components was shown toinhibit both the random (data not shown) anddirectional (chemotaxis) migration of thesecells. Moreover, cigarette smoke componentswere shown not to affect the chemotactic agent.Activated, autologous serum, treated with in-hibitory concentrations of cigarette smoke com-ponents and subsequently dialyzed, was shownto be as effective a chemotactic stimulus asuntreated, activated serum (data not shown).A comparison ofthe concentration ofa partic-

ular constituent in an inhibitory level of ciga-rette smoke with that found to suppress chemo-taxis to 10% of the control value is shown inTable 5. This comparison is based upon theknown concentrations of these constituents incigarette smoke (4, 21, 22) and the observationthat 35 ml of whole cigarette smoke suppressedPMN chemotaxis to 10% of the control level(10). No single agent, at a concentration equiv-alent to that found in 35 ml of whole cigarettesmoke, was found to suppress PMN chemotaxisto 10% of the control level. However, the inhibi-tory concentrations of the unsaturated alde-hydes containing a conjugated double bond(i.e., acrolein and crotonaldehyde) compared

TABLE 5. Comparison of concentration ofcomponents in cigarette smoke with amount needed

for >90% suppression ofPMN chemotaxis

Component

NicotineH2S (Na2S)'YHCN (KCN)'AcetaldehydeAcroleinPropionaldehydeCrotonaldehydeButyraldehydeFurfural

Concn in 35 mlof cigarettesmokeo(mg)

0.220.00270.04180.07340.00360.00970.00140.00070.7

Concn for >90%suppression of

chemotaxisb(mg)

5.021.571.141.5-150.0091.75-17.50.0233.5-353.75-37.5

" Concentration of each cigarette smoke compo-nent in 35 ml of whole cigarette smoke per 5 ml ofPMN suspension previously shown to suppresschemotaxis by >90% (10).

b Concentration of cigarette smoke componentthat suppressed PMN chemotaxis by >90% is ex-pressed in terms of milligrams per 5 ml of PMNsuspension.

' Sulfide and cyanide were added as the sodiumand potassium salts, respectively.

INFECT. IMMUN.

more favorably with their concentration in 35ml of whole tobacco smoke than did the concen-trations of the other components tested. In com-parison to these unsaturated aldehydes, thecorresponding saturated, aliphatic aldehydes(i.e., propionaldehyde and butyraldehyde) weremuch less effective as inhibitors of PMN chem-otaxis. A greater toxicity of the unsaturatedaldehydes relative to their saturated analogueshas been similarly described using cultured as-cites sarcoma cells (24).The low concentrations of acrolein and cro-

tonaldehyde necessary to inhibit PMN chemo-taxis did not cause a detectable loss of PMNintegrity, and their effects were prevented bythe prior addition of cysteine. Thus, these datasuggest that the inhibitory properties of ciga-rette smoke may be largely attributable tothese unsaturated aldehydes, since they mim-icked whole cigarette smoke in their effects onPMN and were inhibitory at low concentra-tions. Furthermore, the protective action ofcys-teine against the inhibitory effects of acroleinand crotonaldehyde suggests that these compo-nents inhibit chemotaxis by their action as oxi-dants and/or thiol-reactive substances. Acro-lein and crotonaldehyde have been described asthiol-alkylating agents (13). A similar thiol-alkylating agent, iodoacetate, was previouslyshown to inhibit PMN chemotaxis, presumablyby inhibiting glucose metabolism (17). Sincethe inhibition of PMN chemotaxis by acroleinand crotonaldehyde occurs without affectingglucose metabolism, these data might suggestthat PMN chemotaxis is dependent upon par-ticular cellular proteins containing one or moreessential thiol group(s) but unrelated to glucosemetabolism. The particular protein(s) affectedand its function in the chemotactic process areyet to be determined. The proteins affected maybe involved with microtubule or microfilamentformation, since the formation ofthese contrac-tile elements has been associated with PMNchemotaxis (2, 7, 16).The more limited protection by cysteine

against the inhibitory effects of whole tobaccosmoke (10) suggests that thiol deactivation isnot exclusively responsible for the overall inhi-bition by whole smoke. Thus, other constitu-ents of cigarette smoke must be considered aspotential inhibitors, acting by a mechanismother than sulfhydryl inactivation. In this con-text, the comparison of inhibitory concentra-tions of nicotine, cyanide, acetaldehyde, andfurfural with their concentration in an inhibi-tory level of cigarette smoke suggests thatthese components may contribute to the totalinhibitory capacity ofcigarette smoke. Further,cysteine (10 mM) did not protect PMNs from


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the deleterious effects of these compounds.However, the relatively high concentrations ofthese compounds necessary for chemotaxis in-hibition and the corresponding loss in cellularviabilities may suggest that the observed ef-fects are at least partially due to cytotoxicity.Of the constituents tested, cyanide deserves

special consideration, since only cyanide signif-icantly affected PMN glucose metabolism. Cya-nide at a concentration inhibitory to chemo-taxis stimulated glycolysis (twofold) and hexosemonophosphate activity (fourfold). Thus, cya-nide in cigarette smoke may be at least par-tially responsible for the metabolic stimulationobserved after exposure of PMNs to wholesmoke, gas phase of smoke, or water-solublefraction (10). The mechanism whereby cyanidestimulates PMN glucose metabolism via glycol-ysis is yet to be determined. However, cyanide(1 mM) has previously been shown to stimulate(55%) the release of 14CO2 from [1-_4C]glucose bynormal leukocytes (19). The cyanide stimula-tion of glucose Cl oxidation was attributed, atleast partially, to an inhibition of myeloperoxi-dase, since cyanide had a much less dramaticeffect on glucose Cl oxidation by myeloperoxi-dase-deficient leukocytes (19). It is unlikelythat cyanide affects PMN glucose metabolismby inhibiting oxidative metabolism, sincePMNs do not appreciably utilize these oxidativepathways for energy generation. Parantheti-cally, the observed inhibition of PMN chemo-taxis by cyanide (10 mM) is consistent with theobservations of Delaunay et al. (12) but is incontrast with those of Lotz and Harris (20).These discrepancies are probably attributableto differences in the methods used to measurechemotaxis.

Finally, sulfide, propionaldehyde, butyralde-hyde, and the phenols can probably be consid-ered the least effective inhibitors ofPMN chem-otaxis in relation to the other, tested compo-nents of cigarette smoke. Inhibition of chemo-taxis by these compounds occurred only at rela-tively high concentrations, which also resultedin dramatic decreases in cellular viabilities.Thus, the chemotaxis of human PMNs has

been shown in this study as well as the previousstudy (10) to be a particularly sensitive bioas-say system for the deleterious agents of ciga-rette smoke. The approach used in the presentstudy was to assess particular components ofcigarette smoke and their effects on PMNchemotaxis. The rationale for this approachwas that the ultimate effects of complex mix-tures (whole cigarette smoke) might be betterunderstood by a study of the effects of individ-ual components. The study ofindividual compo-nents could, for example, lead to a better under-

standing of the distinct biochemical cell func-tions of PMNs that are altered by particularcigarette smoke constituents.

ACKNOWLEDGMENTSWe thank Kerry Bemis for statistical evaluation of the

results and I. Roszman and R. Calmes for critical review ofthe manuscript. We also thank R. H. French for technicalassistance and Debi Walton for her excellent assistance intyping this manuscript.

This investigation was supported by University of Ken-tucky Tobacco and Health Research Institute Project no.KTRB 25096 and 25119.

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