INFECTION AND IMMUNITY, Jan. 1985, p. 169-175 Vol. 47, No. 10019-9567/85/010169-07$02.00/0Copyright C 1985, American Society for Microbiology

Defective Neutrophil and Monocyte Motility in Patients with EarlyOnset Periodontitis

ROY C. PAGE,',3* TOM J. SIMS,' FRANCIS GEISSLER,' LEONARD C. ALTMAN,' AND DAVID A. BAAB2Center for Research in Oral Biology,' Department of Periodontics, School of Dentistry,' and Department of Pathology,

School of Medicine,3 University of Washington, Seattle, Washington 98195

Received 14 May 1984/Accepted 1 October 1984

Several studies have documented suppressed polymorphonuclear neutrophil (PMN) chemotaxis in mostpatients with juvenile periodontitis. In contrast, data regarding PMN chemotaxis in patients with rapidlyprogressive periodontitis are very limited, and monocyte (MN) chemotaxis and random migration of PMNs orMNs from these patients have not been studied previously. Accordingly, we examined cell motility of PMNs andMNs from 27 patients with rapidly progressive periodontitis, 5 patients with juvenile periodontitis, and 37normal control subjects by using a microchamber technique and the synthetic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) as the chemoattractant. As a group, PMNs and MNs from patients with rapidlyprogressive periodontitis manifested significantly enhanced random migration relative to control cells (P <0.001), suppressed directed migration (chemotaxis) at FMLP doses of 10-9 and 10-8 M (P < 0.05), andenhanced directed migration at a dose of 10-6 M FMLP (P < 0.01). In contrast, PMNs from patients withjuvenile periodontitis exhibited normal random migration, and directed migration was significantly suppressedat all doses of FMLP tested (P < 0.05). An abnormality of either PMN or MN motility was observed in 26 of27 patients with rapidly progressive periodontitis. Enhanced random migration was seen in PMNs in 63%,MNs in 39%, and both cell types in 26% of the patients. Suppressed chemotaxis was seen in PMNs in 85%, inMNs in 74%, and in both cell types in 69% of the patients. The prevalence and magnitude of abnormalities inmotility were somewhat lower in treated than in untreated patients. Thus, most, if not all, of this subgroup ofpatients with early onset, highly destructive periodontitis have abnormalities in PMN or MN motility, and thesedefects may differ from those seen in cells from patients with the juvenile form of the disease.

Periodontitis is the most common cause of tooth loss inadult humans. The disease is caused by bacterial coloniza-tion of the surfaces of the teeth in the region of the gingivalsulcus and extension of microbial plaque apically. Bacteriainsinuate themselves between the gingival tissue and theroot surface to cause periodontal pocket formation, exten-sive inflammation, and destruction of the soft tissues andbone housing the roots of the teeth. Periodontitis occurs inmore than one form. Early onset, severe forms which arehighly destructive to the tissues around the teeth are seen insome young children, teenagers, and young adults. Theseforms have been designated as prepubertal, juvenile (JP),and rapidly progressive (RP) periodontitis, respectively (11,12, 14). A more commonly occurring form, usually referredto as adult periodontitis, manifests a later onset, usuallyduring the fourth or fifth decade of life, and has a muchslower rate of progression (14).Although numerous host defense mechanisms are called

into action by the bacterial onslaught at the gingival sulcus,substantial evidence indicates that the phagocytic cells,specifically the polymorphonuclear neutrophilic granulo-cytes (PMNs) and monocytes (MNs), constitute a mostimportant pathway for defense of this area (13). Functionaldefects in the phagocytic cells appear to predispose individ-uals having them to the development of early onset severeperiodontitis; moreover, several recent reports demontratethat patients with these forms of periodontitis manifest ahigh frequency of defects ofPMN chemotaxis as assessed invitro. Lavine et al. (Program Abstr. Annu. Meet. Int. Assoc.Dent. Res., abstr. no. 603, 1976), who used the Boydenchamber technique and a variety of chemoattractants, were

* Corresponding author.

the first to report significant supression of PMN chemotaxisin patients with JP. Their observations were confirmed andextended to include additional patient groups in severalsubsequent publications (3, 5, 7, 18, 20).We have extended the previous studies with a group of 27

patients diagnosed as having RP and 37 normal controlsubjects. A small group of five patients with JP was used forcomparison. Our data demonstrate that relative to cells fromcontrol subjects, leukocytes from RP patients generallymanifest significantly enhanced random migration, sup-pressed chemotaxis at low concentrations of chemoattract-ant, and in some cases enhanced chemotaxis at high concen-trations. Defects were observed in both PMNs and MNs,and the prevalence of defects was much greater than thatpreviously reported.

MATERIALS AND METHODS

Patient selection and processing. Thorough medical anddental histories were taken from all patients and controlsubjects. Criteria for exclusion included systemic illnesseslikely to affect periodontal status, such as diabetes mellitus,chronic ingestion of drugs, including antiinflammatoryagents, antibiotics in the past month, previous periodontaltherapy other than routine tooth cleaning (for untreatedpatients only), and a lack of a desire for participate. A full setof periapical radiographs was obtained for all subjects, and aclinical examination was performed. The examination con-sisted of visual assessment of gingival inflammation, record-ing of missing teeth, and notation of probing depths, using afine probe at six sites around molars and four sites around theremaining teeth. Alveolar bone destruction was assessed onthe radiographs by the method of Schei et al. (15), and theplaque index (9) and gingiva index (17) were recorded. These

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170 PAGE ET AL.

data were used only to establish a diagnosis, and they are notpresented in the present paper. Control subjects had noclinical manifestations of periodontitis, no history of peri-odontal therapy other than routine tooth cleaning, and noprobing depths greater than 4 mm. Patients were assigned adiagnosis of JP or RP on the basis of previously publishedcriteria (14). For RP, these included an age of onset docu-mented by history or previous dental records between pu-berty and age 35 years, lesions generalized without anyconsistent pattern of distribution and affecting more than halfthe teeth, and evidence either historical or radiographic ofrapid periodontal destruction. A diagnosis of JP was assignedto patients between the ages of 13 and 22 years in whom thebone destruction and pocket formation were severe butconfined to the first permanent molars or incisors.

Patients were designated as treated or untreated. Sixteenuntreated patients had had no previous therapy other thanroutine tooth cleaning. Eleven treated patients had beentreated by root planing and root curettage followed by flapsurgery and had been recalled regularly 3 to 6 months aftercompletion of active therapy. In some cases, a single courseof tetracycline at a dose of 1 g/day and lasting 6 days hadbeen administered at the beginning of therapy. We alsotested 5 patients with JP and 37 normal control individuals.

Leukocyte preparation and testing. Approximately 60 ml ofvenous blood was drawn from both patients and healthydonors, using 2% potassium EDTA (final concentration) asanticoagulant. Plasma was removed by centrifugation of thewhole blood at 110 x g for 10 min. The residual blood wasthen diluted with equal amounts of Dulbecco phosphate-buff-ered saline without calcium or magnesium (PBS) (pH 7.2)containing 2% bovine serum albumin fraction V (BSA)(GIBCO Laboratories, Grand Island, N.Y.); 40 ml of dilutedblood was layered over 10 ml of lymphocyte separationmedium (Bionetics, Rockville, Md.) and centrifuged at 900 xg for 30 min. Mononuclear cells were removed and washedthree times with 2% BSA PBS. The cell pellets were thensuspended in 1 ml of 2% BSA PBS to which 51Cr was addedto a final concentration of 200 ,uCi/ml (New England NuclearCorp., Boston, Mass.) (specific activity, 300 to 600 mCi of Crper mg). After removal of the mononuclear cells, the PMNswere isolated from all blood samples by dextran sedimenta-tion and hypotonic saline lysis (4, 6). PMNs were washedthree times with 2% BSA PBS, and 51Cr was added to a finalconcentration of 100 ,uCi/ml. Next, the cells were incubatedfor 45 min at 37°C in humidified incubators containing 95%air and 5% CO2 and then were washed three times with 2%BSA PBS. Cells labeled under these conditions typicallycontained 2 to 3 cpm per 100 cells. The PMN preparationswere greater than 95% pure, as determined by Wright stain,and the mononuclear cell preparations contained 25.9 +7.4% MNs, as revealed by a-naphthyl acetate esterase stain.The mononuclear cells were suspended to a final concentra-tion of 4 x 106 cells per ml, and the PMNs were suspendedto 2 x 106 cells per ml in 2% BSA PBS (pH 7.2) containing0.01% each of MgCl2 and CaCl2 and 0.1% D-glucose. N-For-mylmethionyl-leucyl-phenylalanine (FMLP; Sigma Chemi-cal Co., St. Louis, Mo.) at concentrations from 10-9 to 10-6M was used as the chemoattractant.

Chemotaxis chambers and protocol. Cell migration wasassayed in 48-well microchemotaxis chambers (Neuroprobe,Inc., Cabin John, Md.) as described by Sims et al. (T. J.Sims, F. T. Geissler, and R. C. Page, submitted for publi-cation). The lower attractant-containing compartments wereseparated from the upper cell-containing compartments bytwo polycarbonate filters, 10 ,um in thickness with 3-,um

pores, placed on top of one 100-p.m-thick nitrocellulosefilter, also with 3-p.m pores (Neuroprobe, Inc.), above asilicone gasket. Assembled chambers containing attractantwere warmed to 37°C, loaded with 50 p.l of 51Cr-labeledleukocytes per chamber, and placed in a humidified 5% CO2incubator at 37°C. Mononuclear cells were incubated for 90min, and PMNs were incubated for 60 min. After incubation,the chambers were disassembled, both polycarbonate filterswere discarded, and the nitrocellulose filters were removedand washed twice in large volumes of PBS to remove anyextracellular 51Cr. The filter areas corresponding to individ-ual compartments were easily visible upon drying, allowingthe nitrocellulose filters to be cut into 48 squares, eachcontaining the area above one lower compartment. Theradioactivity of each piece was then rmeasured with a gammacounter. Cells which had dropped off the filter into theattractant well were collected with detergent solution, andtheir 51Cr activity was measured and added to that of thecorresponding filter square.Normal subjects and patients were tested concurrently,

and all subjects were tested on three or more occasions byusing four chambers each time so that the 51Cr counts on atleast 12 lower filters were measured to find the meanmigration rate of the cells of each subject under eachexperimental condition. The 51Cr counts from the gammacounter were recorded magnetically and transferred to amicrocomputer system in which they were normalized tocorrect for variation in labeling efficiency from one cellpreparation to another and, in the case of mononuclear cells,to correct for variation in the percentage of MNs from oneblood donor to another. Experimental minus control, experi-mental divided by control, and the percentage of cells thatmigrated through the upper filters were also calculated andtransmitted, along with the normalized counts, to the Uni-versity of Washington's Locke Computer system for analy-sis by SPSS (Statistical Package for the Social Sciences).The mean of each of these variables for each individual andeach patient population pool was compared with that of thenormal subject pool by using Student's t test (separatevariance estimate), in which mean differences with P < 0.05were considered significant. The significance of the differ-ence between the percentages of normal subjects and ofpatients manifesting an abnormality in chemotaxis or ran-dom motility relative to the normal pool was determined bythe Z-test.

RESULTSThe results of leukocyte-motility studies of cells from

treated and untreated RP patients and normal control sub-jects, presented as the mean and standard error of normal-ized counts versus concentration of FMLP, are shown inFig. 1 for PMNs and in Fig. 2 for MNs. The responsivenessof control donor PMNs increased in a dose-dependentmanner from 10-9 M FMLP to reach a maximum at io-7 Mand then decreased significantly at 10-6 M. A very similarcurve was observed for MNs, except that maximum respon-siveness was observed at 10-9 and 10-8 M FMLP withdecreased responsiveness at 10-7 and 10-6 M. The motilityof patient PMNs differed from control cells in three ways.Patient cells manifested significantly enhanced random mi-gration (migration in cultures containing no chemoattract-ant), suppressed chemotaxis at concentrations of 10-9 and108 M FMLP (P < 0.05), and enhanced chemotaxis at 106M FMLP (P <0.05). In addition, no maximum or plateau ofresponsiveness was reached with patient cells at any FMLPconcentration used. Responsiveness of patient MNs was

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PMN AND MN MOTILITY 171

10-

8-

I-I

a.U~~~~~~~

G /

N

0z 4

0 10-9 l0-8 I0-7 lo-6[CHEMOATTRACTANT]

FIG. 1. Motility of PMNS from 37 normal subjects (0) and 27 RPpatients (0) at various FMLP concentrations.

very similar to that for PMNs except that although it wasgreater than that of control cells at 10-6 M FMLP, thedifference was not statistically significant.

Figure 3 presents data on PMN chemotaxis for JP patientsand for normal subjects. The curve for the normal subjectsdiffers somewhat from that seen in Fig. 1 because the samplesize was smaller, and the subjects were tested at the time theJP patients were tested. The results for JP patients differconsiderably from those obtained for patients with RP. Ourobservations confirm those of Van Dyke et al. (21), in thatrandom migration of JP patient cells did not differ from thatof normal cells. The curve for patient cells paralleled that of

14-

12 :

CL

10

LiJ

0z 8

a

6-~~~~~~4

0 ,

01,~~-,"______0

0 104 iO 1O7 io0[CHEMOATTRACTANT]

FIG. 3. Motility of PMNs from 18 normal subjects (0) and 5 JPpatients (0) at various FMLP concentrations.

normal control cells at all doses of chemoattractant tested,with both reaching a maximum at 10-7 M. However, at allconcentrations the response of patient cells was reduced.To learn whether cell responsiveness was affected by

treatment, the data for RP patients were subdivided andplotted separately for treated and untreated patients. Asseen in Fig. 4, the responses of PMNs from treated anduntreated patients did not differ significantly one from theother or from the curve for the total patient pool (Fig. 1). The

10-

8-

Cl40

0

z4-

10i9 10-8[CHEMOATTRACTANT]

FIG. 2. Motility of MNs from 37 normal subjects (0) and 27 RPpatients (0) at various FMLP concentrations.

0 10-9 lo-8 10-7[CHEMOATTRACTANT]

FIG. 4. Motility of PMNs from 11 treated (0) and 16 untreatedRP patients (0) at various FMLP concentrations.

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172 PAGE ET AL.

LL 0109 l- 107 l6

N

0z

0 ic-9 10-8 o7 106

[CHEMOATTRACTANT]

FIG. 5. Motility ofMNs from 11 treated (0) and 16 untreated RPpatients (0) at various FMLP concentrations.

same can be said for plots of the MN data (Fig. 5), exceptthat the value at 10-6 M FMLP was significantly higher forthe untreated than for the treated group.

During the course of this study we used many methods toanalyze our data. We learned that the manner in whichchemotaxis data are processed and displayed has verymarked effects on the conclusions that can be drawn. Inmost studies of chemotaxis of PMNs from periodontitispatients, the data have been corrected for backgroundrandom migration by calculation of experimental valuesminus control, or experimental values divided by control.When our data are expressed as experimental divided bycontrol (Fig. 6 and 7), the picture which emerges is quitedifferent from that seen when the data are presented in termsof normalized counts which have not been corrected forbackground migration (Fig. 1 and 2). The fact that differ-ences in random migration between control and patient cellsare observed indicates that chemotaxis data corrected forbackground may reflect both random and directed migrationdefects. It is clear that correcting for background migrationmay cause an increase in the proportion of patients that willappear to have a chemotaxis abnormality. An almost iden-tical pattern is seen when the data are calculated and plottedas experimental minus control (data not shown).

Pooling the data for all patients and for normal controlsubjects as has been done in the foregoing graphs shows theoverall trends within each population but tends to obscurethe fact that there is a wide range in the chemotacticresponsiveness of both control and patient cells. Therefore,the data are presented in Fig. 8 and 9 for each control subjectand each treated and untreated patient, together with themean and standard error values. The range covered by onestandard deviation for control cells is also indicated. At allchemoattractant doses, the control PMNs and MNs manifestan extraordinarily wide range of responsiveness. As demon-strated by the other displays of the data, responsiveness ofpatient PMNs and MNs differs significantly from that of

0

z030

z

2- ~ ~ ~ ~ -

a-x

0 io09 10i8 10-7 lo6[CHEMOATTRACTANT]

FIG. 6. Motility of PMNs from 37 normal subjects (O) and 27 RPpatients (0) at various FMLP concentrations presented as experi-mental divided by control counts.

normal cells at all FMLP concentrations except 10-6 M.However, there is overlap, and some values for patient cellsfall within the one standard deviation from the mean forcontrol cells. Because of the apparent wide range of biolog-ical variation in cell motility among control subjects andpatients and the overlap in responsiveness between patientand control cells, making a definitive decision as to whethera given patient is biologically normnal or abnormal is exceed-ingly difficult, even though a high proportion of patients canbe distinguished from the normal population statistically.

Because of the wide range of variation observed in the cellmotility data for both patient and control cells, we haveattempted to achieve greater accuracy by using computerprograms which permitted comparison of data from repeatedtests of each control subject and each patient with the pool

3-

-J0

z00N.

zw

ar-x

0 10- lo-8 10-7 lo-6[CHEMOATTRACTANT]

FIG. 7. Motility of MNs from 37 normal subjects (0) and 27 RPpatients (0) at various FMLP concentrations presented as experi-mental divided by control counts.

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PMN AND MN MOTILITY 173

10l

6

0

z0

4-

4

z

x2

*% A

I :A

* 0*0

P

10-9

*

*S.bA'

I P.1"

P

10

'A

0

*9

I *01,A

.1-1.11

%* O&O.". .0&.4f -- ow--0 0 &

i!tO&0le

TABLE 1. Percentage of subjects in each group at each dose ofFMLP tested manifesting abnormally low chemotaxis'

% Manifesting:Subject group PMNs MNs

10-9b 10-8 10-7 10-6 10-9 10-8 10-7 10-6

Control 3 7 14 10 0 4 38 23Pooled RP 77 81 70 29 56 65 49 13Untreated RP 81 88 81 25 67 75 67 16Treated RP 64 64 55 36 45 54 73 9

' Abnormality was defined as any case in which the mean experimentalcount was significantly lower than the normal pool mean at the indicatedchemoattractant dose at P < 0.05.

b FMLP doses are molar concentrations.

of 10-6 M. The percentage of patients manifesting inhibitedMN chemotaxis over a concentration range of 10-9 to 10-8M FMLP concentration ranged from 56 to 65% for all RPpatients, 67 to 75% for untreated patients, and 45 to 54% fortreated patients. These values were significantly differentfrom controls at P < 0.001. Here, too, the percentagesdropped precipitously to within the range for normal controlsub ects at 10-7 and 10-6 M FMLP. Thus, for both PMN andMN chemotaxis, concentrations of FMLP of 10-9 and 10-8M discriminate between patients and control subjects betterthan do higher concentrations.The frequency of abnormalities in random migration and

chemotaxis is shown by cell type for control subjects andpatients in Table 2. Enhanced random migration of controlcells was seen at frequencies of 10, 15, and 0% for PMNs,MNs, and both cell types, respectively. In contrast, thefrequencies for all RP patients were 63, 39, and 26% forPMNs, MNs, and both cell types, respectively; these differ-ences from control values were valid at the P < 0.001 level.When the pooled data were subdivided, frequencies wereincreased to even higher levels for untreated patients, al-though lower frequencies were observed for the treatedpatients, indicating that treatment may in fact affect PMNand MN motility. Depressed PMN chemotaxis was observedin only 5% of the control population, and none of thesub ects in this group manifested depression of MN or bothMN and PMN chemotaxis. Frequencies were much higher inthe RP patient population, with depressed chemotaxis ob-served in 94, 92, and 92% of PMNs, MNs, and both cells,respectively, for untreated patients (P < 0.001). Frequencieswere somewhat lower for the pooled data and still lowerwhen treated patients were considered separately.

TABLE 2. Frequency of abnormalities in motility by cell type% with:

Enhanced random Abnormal directedSubject group migration' migrationbPMNs MNs Both PMNs MNs Both

Normal 19 15 0 5 0 0Pooled RP 63 39 26 85 74 69Untreated RP 69 58 31 94 92 92Treated RP 55 18 9 73 55 45

a Calculated as normalized counts per minute. Criterion for abnormalitywas P < 0.05 (Z-test).

b Calculated as experimental values divided by control values. Critefion forabnormality was inhibition of chemotaxis at at least two of three doses in therange of 10-9 to 10-7 M FMLP with P < 0.05 (Z-test).

VOL. 47, 1985

0 00 &11

--------a------------0

e.00000

1 4.1 to0

"AIl.p---------9---,

0

&O oA

p

lo-7 10 @

(CHEMOATTRACTANT]FIG. 8. Scattergram of the mean chemotactic responsiveness of

PMNs for each of 37 control subjects (0), 16 untreated RP patients,(0), and 11 treated RP patients (A). Horizontal lines indicate thelimits of one standard deviation of the mean.

of data for all control subjects. Calculations were done ateach concentration of chemoattractant tested to determinewhich concentration or concentrations provide the bestdiscrimination between the normal subject pool and individ-ual patients. Inhibited chemotaxis (P < 0.05) was observedin 3 to 14% of normal control subjects for PMNs and in 0 to38% for MNs (Table 1). For both cell types, the proportionof control subjects manifesting inhibition was much greaterat FMLP concentrations of 10-7 and 10-6M than at 10-1 and10-9 M. The percentage of patients manifesting inhibitedPMN chemotaxis over the concentration range of 10-9 to10-7 M ranged from 70 to 81% for all RP patients, from 81 to88% for untreated patients, and from 55 to 64% for treatedpatients. These values differed from those for normal sub-jects at the level of P < 0.01 to P < 0.001. The percentageswere much lower for all three groups at FMLP concentration

tr

z0

J

z

LJ

x

A

A

0

0*0

* I ..-1 4,I'll i:O

*:o OA0O---

N p

10--s. ----I[CHEMOATTRACTANT]

FIG. 9. Scattergram of the mean chemotactic responsiveness ofMNs for each of 37 control subjects (0), 16 untreated RP patients(0), and 11 treated RP patients (A). Horizontal lines indicate thelimits of one standard deviation of the mean.

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174 PAGE ET AL.

DISCUSSION

The observation by Lavine et al. (Program Abstr. Annu.Meet. IADR, abstr. no. 603, 1976) that PMNs from patientswith JP manifest suppressed or inhibited chemotactic respon-siveness when assessed in vitro has been confirmed andextended by numerous investigators (3, 5, 7, 18, 20). Al-though the cells are abnormal with regard to chemotaxis,they manifest normal deformability, chemokinesis, and ran-dom migration, and they have slightly enhanced adherence(7, 21). Using radioactively labeled FMLP and C5a peptides,Van Dyke et al. (22) have demonstrated membrane receptorsof normal affinity but abnormally low density on PMNs fromJP patients. Whether phagocytosis by these cells is abnormalremains controversial (3, 8). Monocytes from JP patientshave not been well studied; Cianciola et al. (3) reported MNsfrom JP patients to be chemotactically normal, but otherinvestigators have reported otherwise (1, 7).Only very limited studies have been performed on leuko-

cyte chemotaxis with cells from patients with types of earlyonset severe periodontitis other than JP. Bowen et al. (2) andPage et al. (12) assessed PMN and MN chemotaxis inchildren with prepubertal periodontitis. In all cases studied,chemotaxis of either PMNs and MNs or both cell types wassignificantly suppressed. In contrast to cells from patientswith JP, PMNs from two of these children displayed anormal number and affinity of FMLP receptors, but the cellswere unable to adhere to surfaces and therefore unable toperform directed motion. The defect in adherence wastraced to the absence from the cell surface of a specificglycoprotein. Lavine et al. (8) studied PMNs from a group ofpatients ranging in age from 30 to 60 years with "aggressiveperiodontitis," and Van Dyke et al. (20) studied a group ofadults with periodontitis, described as having severe alveo-lar bone loss and ranging in age from 28 to 62 years. In bothcases, abnormalities in chemotaxis were reported.The main thrust of our studies has been to examine PMN

and MN motility in a group of patients with RP. We haveincluded a smaller group of patients with JP for comparativepurposes. Our data demonstrate that RP patients have a veryhigh prevalence of abnormalities in leukocyte motility. In-deed, we failed to detect an abnormality in only 1 of 27 RPpatients. The abnormalities included enhanced random mi-gration, suppressed directed migration (chemotaxis) at lowconcentrations of FMLP (10-9 and 10-8 M), and enhancedchemotaxis at a high FMLP concentration (10-6 M). Al-though control PMNs and MNs manifested maximum che-motactic responsiveness at 10-9 and 10-8 M FMLP, nomaximum was seen at any FMLP concentration used for RPpatient cells. These results contrast with those obtained withPMNs from JP patients, in which random migration wasnormal and maximum responsiveness was reached at 10-7 MFMLP for both control and patient cells, indicating that thebasic mechanisms in leukocyte dysfunction may differ in RPand JP patients. Our observation that random migration forJP PMNs is normal confirms the observation of Van Dyke etal. (19).The prevalence of leukocyte motility defects reported by

us is much higher than that reported by previous investiga-tors studying similar, although not identical, patient groups.For example, Lavine et al. (8) found chemotaxis abnormal-ities in 48% of patients with "aggressive periodontitis," andVan Dyke et al. (20) found enhanced chemotaxis in 10patients and suppressed chemotaxis in 2 of 23 adults withsevere alveolar bone loss. In contrast, we found defects in 26of 27 patients. There are several possible reasons for this.

Our patient group was probably more homogenous thangroups in previous studies, in that the patients were carefullydocumented to have or have had RP with onset betweenpuberty and 35 years of age. We tested both PMNs and MNsover a range of FMLP concentrations and measured bothrandom migration and chemotaxis, whereas most previousinvestigators only tested PMNs and looked only at directedmigration and in some cases used a single concentration ofchemoattractant.A direct comparison of cell motility data for treated and

untreated patients in terms of normalized counts or ofexperimental divided by control values failed to demonstratedifferences which were statistically valid (Fig. 4 and 5). Thisobservation is consistent with most (20) but not all previousreports (16). However, when our data were considered interms of the percentage of patients manifesting abnormalchemotaxis at each chemoattractant concentration tested(Table 1) and the frequency of abnormalities in motility bycell type (Table 2), a somewhat different picture emerged.Without exception, the proportion of patients manifestingabnormalities was greater, in some cases very much so, inuntreated than in treated cases, but these differences werenot statistically significant. However, this trend raises thepossibility that treatment effects may in fact exist. Data fromother laboratories support this contention. Shurin et al. (16)reported marked suppression ofPMN chemotaxis in a youngperiodontitis patient which reverted to normal after treat-ment by extraction of all of the teeth. In the report of VanDyke et al. (20), there appears to have been partial reversionof the chemotaxis abnormality in a small number of treatedpatients. There is additional reason to expect reversion orpartial reversion of the abnormalities after treatment. SomeActinobacillus species, bacteria implicated in the pathogen-esis of early onset periodontitis, produce a leukotoxin whichcan interfere with PMN and MN function (10), and severalputative periodontal pathogens produce substances whichinteract with PMNs and inhibit their chemotactic responsive-ness (19). Thus, whether treatment does in fact affectleukocyte motility as assessed in vitro remains uncertain.Our observations serve to illustrate some of the pitfalls

encountered in assessing cell motility in vitro in patients andcontrol subjects. The range of random and directed cellmigration by leukocytes from normal donors is enormous.Since we tested each subject on at least three separateoccasions with a minimum of four replicates each time, thislarge range of responsiveness most likely results from bio-logical variation, not technical difficulties with the meas-urements. When superimposed on this background, dis-crimination of differences between patient and control cellsbecomes very difficult. Our data show clearly that a singleassessment, even with replicates, at a single concentration ofchemoattractant, especially when done only with PMNs,can be quite misleading. Indeed, cells from a given patientmay show inhibited chemotaxis at one concentration ofattractant, normal values at another, and enhanced chemo-taxis at still another. When dose curves cannot be done,concentrations of FMLP of 10-9 and 10-8 M appear toprovide the best discrimination between normal subjects andpatients. Pooling data can also be misleading. A comparisonof Fig. 6 and 7, in which the data have been pooled andprocessed with n being the number of replicate measure-ments, with Fig. 8 and 9, in which data points for eachpatient are presented separately, results in very differentconclusions. Figures 6 and 7 indicate that differences invalues for patient and control cells are enormous for allFMLP concentrations except 10-6 M and that the popula-

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PMN AND MN MOTILITY 175

tions are totally separated. Figures 8 and 9, on the otherhand, demonstrate that values for many patients fall withinone standard deviation of the normal mean and that there isoverlap. Finally, these and other figures and tables demon-strate that analysis of the data by a single method may leadto erroneous conclusions. Presentation of the data as exper-imental divided by control or experimental minus controlvalues, as has been frequently done, obscures potentialdifferences between patient and control cells with regard torandom migration. In the present study, failure to analyzethe data in terms of percentage of affected and nonaffectedpatients or in frequencies of cell abnormalities would havemasked possible effects of treatment and the unexpectedlyhigh prevalence of abnormalities.

Regardless of the method of data processing and statisticalanalysis used, at some point a decision must be made as towhether cell motility of a given patient is normal or abnor-mal. We believe the criteria we have set are reasonable. Weconsidered random migration to be abnormal when themotility of a given patient's cells differed from that of a poolof normal cells at the P < 0.05 level. We consideredchemotaxis to be abnormal when two of three data points inthe FMLP concentration range of 10-9 to 10-7 M weresignificantly lower than control values at the P < 0.05 level.Although these criteria are fairly rigid, they increased dis-crimination by reducing the number of individuals in thenormal population with apparent cell-motility abnormalities.

ACKNOWLEDGMENTSThis work was supported by Public Health Service grants

DE-02600 and DE-07063 from the National Institutes of Health.

LITERATURE CITED

1. Altman, L. C., R. C. Page, J. L. Ebersole, and G. E. Vandesteen.1982. Assessment of host defenses and serum antibodies tosuspected periodontal pathogens in patients with various typesof periodontitis. J. Periodontal Res. 17:495-497.

2. Bowen, T. J., H. D. Ochs, L. C. Altman, T. H. Price, D. E. VanEpps, D. L. Brautigan, R. E. Rosin, W. D. Perkins, B. M.Babior, S. J. Klebanoff, and R. J. Wedgewood. 1982. Severerecurrent bacterial infections associated with defective adher-ence and chemotaxis in two patients with neutrophils deficientin a cell-associated glycoprotein. J. Pediatr. 101:932-940.

3. Cianciola, L. J., R. J. Genco, M. R. Patters, J. McKenna, andC. J. Van Oss. 1977. Defective polymorphonuclear leukocytefunction in a human periodontal disease. Nature (London)265:445--447.

4. Clark, R. A., and H. R. Kimball. 1971. Defective granulocytechemotaxis in Chediak-Higashi syndrome. J. Clin. Invest.50:2645-2652.

5. Clark, R. A., R. C. Page, and G. Wilde. 1977. Defectiveneutrophil chemotaxis in juvenile periodontitis. Infect. Immun.18:694-700.

6. Gallin, J. I., R. A. Clark, and E. J. Goetzl. 1978. Radioassay ofleukocyte locomotion, p. 79. In J. I. Gallin and P. G. Quie (ed.),Leukocyte chemotaxis: methods, physiology and clinical impli-cations.

7. Genco, R. J., T. E. Van Dyke, B. Park, and H. U. Horoszewicz.1980. Neutrophil chemotaxis impairment in juvenile periodon-titis. Evaluation of specificity adherence, deformability, andserum factors. J. Reticuloendothel. Soc. 28(Suppl.):81s-91s.

8. Lavine, W. S., E. G. Maderazo, J. Stolman, P. A. Ward, R. B.Cogen, I. Greenblatt, and P. B. Robertson. 1979. Impairedneutrophil chemotaxis in patients with juvenile and rapidlyprogressing periodontitis. J. Periodontal Res. 14:10-19.

9. Loe, H., and J. Silness. 1963. Periodontal disease in pregnancy.I. Prevalence and severity. Acta Odontol. Scand. 21:533-551.

10. McArthur, W. P., C.-C. Tsai, P. C. Baehni, R. J. Genco, and N.Taichman. 1981. Leukotoxic effects of Actinobacillus acti-nomycetemcomitans. Modulation by serum components. J.Periodontal Res. 16:159-170.

11. Page, R. C., L. C. Altman, J. L. Ebersole, G. E. Vandesteen,W. H. Dahlberg, B. L. Williams, and S. K. Osterberg. 1983.Rapidly progressive periodontitis: a distinct clinical condition.J. Periodontol. 54:197-209.

12. Page, R. C., T. Bowen, L. C. Altman, G. E. Vandesteen, H.Ochs, P. Mackenzie, S. Osterberg, L. D. Engel, and B. L.Williams. 1983. Prepubertal periodontitis. I. Definition of aclinical disease entity. J. Periodontol. 54:257-271.

13. Page, R. C., and H. E. Schroeder. 1981. Current status of hostresponse in chronic marginal periodontitis. J. Peridontol.52:477-491.

14. Page, R. C., and H. E. Schroeder. 1982. Periodontitis in man andother animals. S. Karger, Basel.

15. Schei, O., J. Waerhaug, A. Lovdal, and A. Arno. 1959. Alveolarbone loss as related to oral hygiene and age. J. Periodontol.30:7-16.

16. Shurin, S. B., S. S. Socransky, E. Sweeney, and T. Stossel. 1979.A neutrophil disorder induced by Capnocytophaga, a dentalmicroorganism. New Engl. J. Med. 301:849-854.

17. Silness, J., and H. Loe. 1964. Periodontal disease in pregnancy.II. Correlation between oral hygiene and oral condition. ActaOdontol. Scand. 22:121-135.

18. Vandesteen, G. E., L. C. Altman, and R. C. Page. 1981.Peripheral blood leukocyte abnormalities and periodontal dis-ease. J. Periodontol. 52:174-180.

19. Van Dyke, T. E., E. Bartholomew, R. J. Genco, J. Slots, andM. J. Levine. 1982. Inhibition of neutrophil chemotaxis bysoluble bacterial products. J. Periodontol. 53:502-508.

20. Van Dyke, T. E., H. U. Horoszewicz, L. J. Cianciola, and R. J.Genco. 1980. Neutrophil chemotaxis dysfunction in humanperiodontitis. Infect. Immun. 27:124-132.

21. Van Dyke, T. E., H. U. Horoszewicz, and R. J. Genco. 1982. Thepolymorphonuclear leukocyte (PMNL) locomoter defect in ju-venile periodontitis. Study of random migration, chemokinesisand chemotaxis. J. Periodontol. 53:682-687.

22. Van Dyke, T. E., M. J. Levine, L. A. Tabak, and R. J. Genco.1981. Reduced chemotactic peptide binding in juvenile periodon-titis: a model for neutrophil function. Biochem. Biophys. Res.Commun. 100:1278-1284.

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