Vol. 35, No. 1INFECTION AND IMMUNITY, Jan. 1982, p. 71-780019-9567/82/010071-08$02.0O/0

Neutrophil Chemotaxis by Propionibacterium acnes Lipaseand Its Inhibition

WEI-LI LEE,'* ALAN R. SHALITA,1 KAMALA SUNTHARALINGAM,2 AND SENIH M. FIKRIG2Department ofDermatology' and Department of Pediatrics,2 State University ofNew York, Downstate

Medical Center, Brooklyn, New York 11203

Received 20 February 1981/Accepted 27 August 1981

The chemoattraction of Propionibacterium acnes lipase for neutrophils and theeffect of lipase inhibitor and two antibiotic agents on the chemotaxis wereevaluated. Of the various fractions tested, partially purified lipase (fraction 2c)was the most active cytotaxin produced by P. acnes. Serum mediators were notrequired for the generation of chemotaxis by lipase in vitro. Diisopropyl phospho-fluoridate at low concentration (10-4 mM) completely inhibited lipase activity aswell as polymorphonuclear leukocyte chemotaxis generated by lipase. Tetracy-cline hydrochloride and erythromycin base at concentrations of 10-1 mM and 1mM, respectively, caused 100%o inhibition of PMN migration toward lipase orzymosan-activated serum. The inhibiting activity of the antibiotics was directedagainst cells independently of any effect on lipase. Chemotaxis by P. acnes lipasesuggests a wider role for this enzyme in the inflammatory process and thepathogenesis of acne vulgaris.

The etiological events which ultimately lead toacne lesions are complex and multifactorial,involving androgenic stimulation of the seba-ceous gland with resulting increased sebum pro-duction, hydrolysis of sebum triglycerides bythe follicular microflora, alterations in follicularepithelial differentiation, and a mixed inflamma-tory infiltrate. The fact that large numbers ofPropionibacterium acnes occur in all stages ofthe acne process and the successful use ofantibacterial therapy in the treatment of acnesuggest that microbial components play an im-portant role in the disease. Lipase, the extracel-lular enzyme derived from P. acnes, is reportedto be responsible for the hydrolysis of sebumtriglycerides to free fatty acids which have beenimplicated as both irritants and comedogenicagents and which lead to an intensification of theinflammatory process (8, 12, 22, 23). Recentreports, however, suggest that the role of thisorganism may be more complicated (13, 24, 31).A macrophage-specific cytotoxin derived fromanaerobic diptheroids has been reported by Wil-kinson et al. (32, 33). It is possible that chemo-taxis may be the mechanism for the initiation ofinflammatory reactions after release of bacterialproducts into the dermis.

In contrast to the wealth of the informationavailable on the composition and profile of tox-ins or allergins of most pathogenic bacteria, thecorresponding data on P. acnes are poorly docu-mented. Research in the immunological aspecthas also been hampered by a lack of purifiedantigens. One of the major criticisms of most

published studies is that the antigen preparationsare usually concentrated culture supernatantsand are complex in nature and of unknowncomposition. It is essential to isolate and ana-lyze well-defined antigens to understand thebiological and immunological roles of this organ-ism. This investigation was undertaken to deter-mine whether purified lipase from P. acnes waschemotactic for neutrophils.

MATERIALS AND METHODSCultivation of microorganism. P. acnes was cultured

as described previously (14). The organism was acti-vated by repeated subculture in the same medium usedfor the large scale batch culture. To assure anaerobio-sis, the broth was gassed with a mixture of N2-CO2(95:5, vol/vol) before inoculation. Organisms weregrown in a shaking metabolic incubator (100 rpm) at35°C for 5 to 6 days. The late log phase culturesupernatants were obtained by centrifugation at 8,000x g for 40 min in a Sorvall RB-2 centrifuge.

Purification of lipase. The preparation of lipase wascarried out by the procedures described by Pablo et al.(17) and Hassing (10), with some modifications. Theseinclude the ultrafiltration with an Amicon hollow fiberconcentrator (model DC2) excluding substances ofmolecular weights below 10,000 and precipitation withabsolute alcohol to 50%6 saturation, followed by gelfiltration on Sephadex G-100, and two ion-exchangechromatographies on DEAE-Sephadex A-50 and CMBio-Gel columns. The Sephadex G-100 chromatogra-phy was performed in distilled water or 20 mM sodiumacetate buffer (pH 5.5). The DEAE-Sephadex chroma-tography was performed by elution with an increasingsalt gradient in phosphate buffer. The subsequent CMBio-Gel chromatography was carried out by using a

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two-step gradient from 0.8 M NaCl in 20 mM sodiumacetate buffer (pH 5.5, 600 ml) to 0.02 M NaCl in thesame buffer (400 ml). The eluents were collected in anLKB automatic fraction collector with a UV monitorscanning at 280 nm and assayed for lipase activity. Thelipolytic fractions were pooled and either dialyzedexhaustively against distilled water or concentratedand desalted in an Amicon ultrafiltration cell (Diafol402) equipped with an XM 50 membrane, lyophilized,and stored at -70°C. All enzyme purification proce-dures were carried out at 4°C.The catalytic activity of the lipase was routinely

assayed with a Radiometer pH-stat using an olive oil-gum arabic emulsion substrate (20). The specific activ-ity of the lipase preparation was expressed in units permilligram of protein. One unit is equivalent to theproduction of 1 ,umol of free fatty acids per min.Protein was determined by the Folin phenol reagentwith bovine serum albumin as the standard (15).

Polyacrylamide-gel electrophoresis. The purity of li-pase was examined on sodium dodecyl sulfate-poly-acrylamide gel electrophoresis in 10o gel by a modifi-cation of the method of Weber and Osborn (28) inwhich 0.1% sodium dodecyl sulfate was used. Lyophi-lized samples (0.02 to 0.05 mg) were dissolved insample solution containing 2-mercaptoethanol incu-bated at 37°C for 1 h and applied to the gel. Electro-phoresis was carried out at 4 mA/tube for 3 to 4 h withbromophenol blue as the marker dye. After the com-pletion of electrophoresis, the gels were first fixed andstained in a solution of 0.2% Coomassie blue-509methanol-5% alcohol and then destained in 7% aceticacid-5% methanol. Molecular weights were deter-mined by comparison with a series of standard pro-teins run under the same condition: transferin (84,000),bovine serum albumin (66,500), ovalbumin (45,000),chymotrypsinogen (25,700), myoglobin (16,900), cyto-chrome c (13,000), and lysozyme (14,400) (28).

Neutrophil chemotaxis. Neutrophil chemotaxis wasassayed in vitro as previously described (5, 7). Briefly,human neutrophils obtained from postpartum bloodwere isolated by a mixture of Methocel-isopaque (3),washed and resuspended in TC 199 (GIBCO Labora-tories) containing 2% bovine serum albumin at a finalconcentration of 2.5 x 106 polymorphonuclear leuko-cytes (PMN)/ml. Samples of the cell suspension werethen placed in the upper compartment of a modifiedBoyden chemotactic chamber separated from a che-motactic or control substance (TC 199) by a 5-pmmembrane filter (Millipore Corp.). After incubation for3 h at 37°C in a humidified atmosphere of5% C02, thefilters were removed and stained, and cell migrationwas quantified. The chemotactic activity was evaluat-ed by taking the average counts in five random high-power fields (HPF) of the number of PMN found onthe lower filter surface. Results of chemotaxis activitywere expressed in PMN/HPF after subtracting ran-dom migration. Duplicate chambers were used foreach material tested. The standard error of the meanfor the duplicate filters was less than 5%. The day today variability between tests sequentially in a singlecontrol ranged between 5 to 10%.

The ability of P. acnes lipase to generate serum-derived chemotactic factors was assayed by incubat-ing various isolated fractions with an equal volume of20%o serum for 30 min at 37°C before adding to thelower compartment. The chemotactic activity of the

partially purified lipase and lipase-generated serumwas tested in a dose-response fashion by either addingvarious amounts of enzyme preparation (fraction 2c) inTC 199 and applying directly to the lower chamber orby incubating the enzyme with an equal volume ofserum for 30 min at 37°C, and TC 199 was used todilute the serum to 10%o before use. Zymosan-activat-ed serum (ZAS) at a concentration of 1 mg/ml wasused as a reference standard. In the inhibition studies,stock solution (0.1 M) of antibiotics and diisopropylphosphofluoridate (DFP) were prepared in TC 199 andisopropyl alcohol. Serial dilutions of these compoundswere made in TC 199. Lipase and ZAS were treatedwith an equal volume of antibiotic or DFP to achievethe appropriate concentration for the lower compart-ment. The mixtures were incubated at 37°C for 30 min,and the residual lipase activity was determined by thestandard pH-stat method. To determine the effect ofthese compounds on chemotaxis, the lipase (0.01 mg/ml), ZAS (1 mg/ml) containing antibiotic, or DFP wasadded directly to the lower compartment unless statedotherwise. Similar measurements were made in filtersobtained from control chambers containing TC 199 (orTC 199-isopropyl alcohol when DFP was used) with orwithout the chemoattractants. The percentage of inhi-bition was calculated by comparison of the chemotac-tic activity obtained with cells migrating towards che-moattractants and toward antibiotic- or DFP-treatedchemoattractants. To study the inhibitory effect ofantibiotic directed against cells, neutrophils (5 x 106cells per ml) were incubated in a shaking water bath at37°C for 30 min in the presence of an equal volume of0.02 mM antibiotics. After incubation, the residualchemotactic responsiveness of the treated cell wastested in chemotactic chambers by using the bacterialfactors in the lower compartment.

RESULTS

Purification of lipase. The ethanol precipitateswere fractionated on Sephadex G-100 by elutingwith water or sodium acetate buffer. In bothsystems it separated to give a peak (fraction la)in the excluded volume and a broad peak con-taining small-molecular-weight polymers andsalts (Fig. 1). Rechromatography (not shown) ona column of Sephadex 200 eluting with the samebuffer gave a single broad peak (a portion ofwhich was in the excluded volume). These re-sults indicate that lipase has a molecular weightin the 150,000 to 200,000 range. The lipolyticactivity was found to be confined to the high-molecular-weight peak and was combined andfractionated on a DEAE A-50 column. Thelipase was not absorbed by the anionic columnand was eluted out before the gradient started.The second cationic chromatographic proffle isshown in Fig. 2. The enzyme assay of thefractions (2a, 2b, and 2c) showed that theyvaried in lipolytic activity, and the fraction thateluted at 0.55 M NaCl contained greater than90% of the total lipase activity.The sodium dodecyl sulfate-polyacrylamide

gel electrophoretic pattern of the purified lipase

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0

0 0.6cr

r-U(nT,)mC-)-4

--

C1

0.(A

FRACTIONFIG. 1. Chromatography on Sephadex G 100 of products obtained by ethanol precipitation of culture

supernatant.

(fraction 2c) is shown in Fig. 3. The majorprotein band appeared homogeneous migratingat a mobility similar to that of the proteinstandard bovine serum albumin with an estimat-ed molecular weight of 65,000 ± 3,000. In addi-tion, one minor protein component with moder-ate mobility of 25,000 was also present.

Neutrophil chemotaxis and inhibition. Frac-tions isolated throughout the lipase purificationprocedure and zymosan were assayed for neu-trophil chemotactic activity (Fig. 4). All frac-tions except fraction 2a, the low salt eluted CMBio-Gel fraction, demonstrated chemotactic ac-tivity. Fraction 2c was the most active cytotaxinamong the fractions. In contrast to zymosan,

which required compiement components for itschemotactic activity, the lipase exerted a directchemotactic effect on PMN in the absence ofserum. The amount of chemotactic activity pro-duced was directly proportional to the amount ofP. acnes lipase added (Fig. 5). When >15 ,ug ofenzyme was used, chemotactic activity pro-duced by lipase fell rather sharply. The effect ofexcess enzyme was qualitatively similar to thatobserved with lipase-generated serum. The pres-ence of serum inhibitor of the P. acnes lipaseprobably accounts for the larger concentrationof enzyme required to generate comparable che-motactic activity in serum.The results of enzyme inhibition by various

i2 a 2b 2c

GRADIENT

1O 30 50 70 90 110 130

FRACTION

FIG. 2. Chromatography of P. acnes lipase on CM Bio-Gel. The dashed line shows the concentration ofNaClin the gradient. The fractions were pooled as indicated by the bar lines.

0.8

0.6

eVi

04

0.2

r--D

m

--i

Ca

100

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74 LEE ET AL.

0

OAO

LcrPASE

IYMO

L

---T------------

,.2 4 :. t, ORELAT VE MOBIL'y

FIG. 3. Sodium dodecyl sulfate (10%) polyacrylamide gel pattern and molecular weight of purified lipase.Arrows indicate the position of protein bands of P. acnes lipase.

compounds are shown in Table 1. It is apparentthat tetracycline and DFP were effective lipaseinhibitors, and the degree of inhibition was con-centration dependent. Completely lipolytic inhi-bition was found at concentrations of 1 mM and10-4 mM for tetracycline and DFP, respective-ly. In contrast, erythromycin demonstrated noeffect upon the lipase. Similar conclusions weremade by Weeks et al. (31), except that the DFPinhibition was found more pronounced withfraction 2c. The effects of these compounds onPMN migration are shown in Fig. 6A and B.

Both antibiotics suppressed the chemotactic ac-tivity of ZAS, and linear inhibition was found atconcentrations between 0.01 and 0.1 mM tetra-cycline, and at concentrations between 0.5 to 1mM erythromycin. The antibiotic inhibition wasnot specific to the chemotactic agent. Paralleldose-response curves were found when eitherlipase or ZAS was used as the chemotacticstimulus. It should be noted, however, that thedegree of inhibition appears to be greater whenlipase is used as the chemoattractant.The effect of DFP on neutrophil chemotaxis

LLa-I

Cf,z

La-

w

z

zwcC

100

80

60

40

0Zy la 2a 2b 2c

(1mg) (0.1mg) (0.1lmg) (0.01 mg)FIG. 4. Neutrophil chemotaxis. Hatched bars represent mean value for neutrophil chemotaxis ±1 standard

deviation when serum is supplemented with bacterial factor. Open bars represent comparable results by addingbacterial factor alone. Values given = PMN migration (TC 199 + chemoattractant) - PMN random migration(TC 199).

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NEUTROPHIL CHEMOTAXIS BY P. ACNES

Li.a.

0100z0.

wco

z50504w

P. ACNES LIPASE (,Ig)

FIG. 5. Chemotactic activity of neutrophils in re-sponse to P. acnes lipase (0) and lipase-generatedserum (0). Negative control activity was 3.5. Positivecontrol (ZAS) activity was 65.4.

(Fig. 6B) was found to be quite different fromthat of the antibiotics. The DFP effect wasspecific for lipase and showed no effect on ZASat concentrations ranging from 10-3 to 10-6 mM.The dose-response curve was characterized by ahigh initial response slope at low concentrationsofDFP. Additional information on whether DFP(10-3 mM) is inhibitory to cell-directed or lipase-directed chemotactic activity was obtained byusing a Bio-Gel P 2 column which excluded DFPfrom DFP-treated lipase. No significant migra-tion occurred when the eluting lipase (0.01 mg/ml) was tested in the lower compartment.These results suggest that the inhibition by

antibiotics appears to be independent of theireffect on the lipase. Further experiments wereconducted to determine whether incubation ofPMN with antibiotics could result in a sup-

1001Z (A)~0/

z50.I

TABLE 1. Inhibition of P. acnes lipase activityCompounda Concn (mM) % Inhibition

Tetracycline 10-2 0lo-0 211 100

Erythromycin 10-' 01 0

Diisopropyl 10-6 38fluorophosphate 10-5 58

10-4 100

a Compounds were incubated at 37°C for 30 minwith the enzyme preparation (2c) before the assay forresidual lipase activity.

pressed chemotactic response. PMN were incu-bated at 37°C for 30 min in TC 199 containing0.01 mM antibiotics. Cells suspended in bovineserum albumin containing TC 199 were eitherwashed or directly placed in the Boyden cham-ber and tested for their chemotactic responsesby using the lipase or ZAS as a chemoattractant.Controls consisted of cells incubated in the samemanner with TC 199. The chemotactic activity(PMN/HPF) was compared to the results ob-tained from the chambers containing cells alone.The antibiotic (0.01 mM) was also placed in thelower compartment to assess if chemotacticactivity was present. Preincubation with antibi-otics caused a 60 and 38% inhibition with tetra-cycline and erythromycin, respectively, whencells were tested against lipase and an inhibitionof 31 and 27%, respectively, when cells weretested against ZAS (Table 2). The concentrationof antibiotic used in this experiment was consid-erably smaller than required to inhibit a similardegree of neutrophil chemotaxis when the anti-biotic was incubated with the chemotactic fac-tor. Since the antibiotic alone showed minimal

(B)

of-

4 0-5 1&-4 g0oINHIBITOR CONCENTRATION (mM)

FIG. 6. Inhibitory effect of antibiotics (A) and DFB (B) on neutrophil chemotaxis. Various concentrations oftetracycline (T), erythromycin (E), and DFP were incubated with lipase (2c) or zymosan activated serum (ZAS)as described in the text. Symbols: 0, T + 2c; A, T + ZAS; 0, E + 2c; A, E + ZAS; *, DFP + 2c; x, DFP +ZAS.

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TABLE 2. Effect of preincubation with antibiotics on subsequent chemotactic activity of human neutrophilsNeutrophil chemotaxis

Test samplea Chemoattractant NMN/HPF hi

%cnemoiaionPMN/HPFb to Inhibition'

Cell lipase 121Cell + T lipase 40 67Cell + E lipase 57 53Cell + T then washed lipase 48 60Cell + E then washed lipase 75 38

Cell ZAS 106Cell + T ZAS 14 87Cell + E ZAS 32 70Cell + T then washed ZAS 73 31Cell + E then washed ZAS 77 27

a Neutrophils were incubated at 37°C for 30 min with 0.01 mM of antibiotic in TC 199. After incubation, cellswere either added directly to the upper compartment of the chemotactic chamber or washed to remove theantibiotic before testing for the chemotactic activity. E, Erythromycin; T, tetracycline.

b Results are shown in PMNtHPF after subtracting random migration in controls.c In this study, duplicate experiments had coefficients of variation ranging between 5 or 10%. All values less

than 10% inhibition would therefore be considered insignificant.

chemotactic activity (5 and 2 PMN/HPF fortetracycline and erythromycin, respectively),the observed suppression in chemotaxis afterpreincubation was not the result of subjectingPMN to the chemotactic factor. It should benoted that when antibiotic was placed with PMNin the upper compartment throughout the assay,the degree of antibiotic inhibition increased sig-nificantly (P < 0.001) when tested against ZAS.

DISCUSSIONThe effects of various bacterial products upon

the induction of immune responses and theirinteraction with leukocytes have been well stud-ied. The anaerobic diphthroid Corynebacteriumparvum, which appears to be the same organismas Propionibacterium acnes and Propionibacter-ium granulosum (4), has excited considerableinterest in recent years because it markedlyinfluences the course of immune responses.Thus, it acts as stimulant of the reticuloendothe-lial system, as an adjuvant to enhance humoraland cell-mediated responses, and as an antitu-mor agent which modifies the immunologicalresponse in tumors (1, 9, 16, 21).We have studied the chemotactic activity of

P. acnes lipase which acts directly toward neu-trophils in the absence of serum. The character-istics of this factor appeared to be similar to themacrophage-specific chemotactic factor report-ed by Wilkinson et al. (33). The production ofboth factors, elaborated at late log phase pla-teaued at 6 to 8 days before leveling off coincid-ed with lipase production and was independentof the active cell growing log phase (3 days).They both are macromolecules and stable toheat (56°C), indicating that the tertiary structure

is probably not involved in its function. Theoriginally reported macrophage-specific chemo-tactic factor now appears to be active for neutro-phils as well (personal communication). Thechemotactic activity of lipase toward other celltypes requires further examination. In agree-ment with its eluting behavior on gel filtration,P. acnes lipase and the majority of microbiallipases have been reported to be large moleculesof aggregates of protein-lipid or protein-protein.A structure of this type makes it possible tosuggest that the heterogeneity observed on CMBio-Gel is due to the presence of moleculeshaving different portions of the same basic struc-ture. It also explains some of the confficting dataon the size and specificity of P. acnes lipase asdescribed by various groups who isolated cellsfrom different growth phases ((H. H. Ku, per-sonal communication) or by various procedures(10, 17). The chemotactic factors described hereapparently differ from those that were reportedby Keller and Sorkin (11) and Ward et al. (19, 26,27) in that they are low molecular weight (150 to1,500 A), their production is related to the activegrowth phase of bacterial replication, and theyare known to act indirectly by activating com-plement or a similar enzyme system in plasma.Similar chemotactic factors are also producedby P. acnes (18, 29, 30).The study dealing with neutrophil inhibition

after treatment with antibiotics or lipase inhibi-tor is of special interest with regard to thepathogenesis of acne vulgaris and confirms simi-lar observations by Esterly et al. (6). The mecha-nism by which antibiotics might affect neutrophilmobility is not clear. Evidence at this timesuggests that a cellular mode of action is at least

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partially responsible for chemotactic inhibition.This conclusion is supported by the results inTable 1 and Table 2 indicating that althougherythromycin does not inactivate the lipolyticactivity of lipase, the preincubation of PMNcontaining the antibiotic results in suppressedchemotactic responsiveness. In addition, thiscell-direct inhibition is not chemotactic factorspecific as they reduced cell mobility towardlipase as well as toward ZAS. Suppression at thecellular level could explain the observed nonspe-cific inhibition of chemotactic agents. However,the greater suppressive effect observed whenantibiotic was present throughout the assay inthe ZAS system may support the presence ofboth cell-direct and factor-direct inhibitor as thelatter was caused after the contact of the cell andZAS.

Diisopropyl phosphofluoridate and other ana-logs of phosphonate ester have been known toirreversibly inhibit the serine esterase of neutro-phils at concentration of10-5 M or less (2, 25).In the present study, DFP has been shown toexhibit a marked inhibitory effect on neutrophilchemotaxis against bacterial lipase. A correla-tion seems to exist between the inhibition ofcatalytic activity and the inhibition of chemotax-is by DFP as the two phenomena are affected bythe same concentration. It is possible that serineresidue on the active site of P. acnes lipase (17)may also be involved in attracting neutrophils.Chemotaxis may be affected by inhibiting ei-

ther the chemotactic factor or the migratingresponse of the cell under study. In this investi-gation we have demonstrated that under ourexperimental conditions the inhibition of neutro-phil chemotaxis by antibiotics is due to a directeffect on the neutrophil rather than on the che-motactic factor. The relatively low concentra-tions required for this inhibition suggest thatantibiotics may play an additional anti-inflam-matory role when used in the treatment of acne.

ACKNOWLEDGMENTThis research was supported in part by a grant from the

Dermatology Foundation.

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2. Becker, E. L. 1971. Biochemical aspects of the polymor-phonuclear response to chemical factors, p. 243-254. InR. Austin and E. L. Becker (ed.), Biochemistry of theacute allergic reactions. Blackweli, Oxford.

3. Boyum, A. 1964. Separation of white blood cells. Nature(London) 204:793-794.

4. Cummins, C. S., and J. L. Johnson. 1974. Corynebacteri-um parvum: a synonym for Propionibacterium acnes. J.Gen. Microbiol. 80:433-442.

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11. Keller, H.V., and E. Sorkin. 1967. Studies on chemotaxis:V. on the chemotactic effect of bacteria. Int. Arch. Allerg.31:505-517.

12. Kirschbaum, J.O., and A. M. Klgman. 1963. The patho-genesis of Corynebacterium acnes in acne vulgaris. Arch.Dermatol. 88:832-833.

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17. Pablo, G., A. Hammons, S. Bradley, and J. E. Fulton.1974. Characteristics of the extracellular lipase fromCorynebacterium acnes and Staphylococcus epidermidis.J. Invest. Dermatol. 63:231-238.

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26. Ward, P. A., C. G. Cochrance, and H. J. Muller-Eber-hard. 1966. Further studies on the chemotactic factor ofcompliment and its formation in vivo. Immunology11:141-153.

27. Ward, P. A., I. H. Lepow, and L. J. Newman. 1968.Bacterial factors chemotactic for polymorphonuclear leu-kocytes. Am. J. Pathol. 52:725-736.

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32. Wilkinson, P. C., G. J. O'Neill, and K. G. Wapshaw. 1972.Role of anaerobic Coryneforms in specific and non-specific immunological reactions. Immunology24:997-1006.

33. Wilkinson, P. C., G. J. O'Neill, and K. Wapshaw. 1973.Role of anaerobic coryneforms in specific and non-specif-ic immunologic ractions. II. production of a chemotacticfactor specific for macrophage. Immunology 24:997-1006.

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